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Stretching fibroblasts remodels fibronectin and alters cancer cell migration.

Ao M, Brewer BM, Yang L, Franco Coronel OE, Hayward SW, Webb DJ, Li D - Sci Rep (2015)

Bottom Line: Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors.Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin.A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN.

ABSTRACT
Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

No MeSH data available.


Related in: MedlinePlus

Stretched NAFs deposit fibronectin in an organized format.(a) NAFs (yellow) were grown in control (unstretched) and stretched devices. (b) Representative phase images of NAFs grown in control or stretched devices (after stretching), illustrating the random orientation of the cells attached to the PDMS membrane (scale bar is 50 μm). (c) Characteristic images demonstrating the immunostaining of fibronectin produced by unstretched NAFs, stretched NAFs, as well as CAFs (scale bar is 50 μm). (d) Average angles between fibronectin fibers measured as reported56 to compare fibronectin alignment. Both CAFs and stretched NAFs produced fibronectin with smaller angles than the fibronectin produced by unstretched NAFs. Error bars represent S.E.M for 50 cells from three individual experiments (*p < 0.002).
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f3: Stretched NAFs deposit fibronectin in an organized format.(a) NAFs (yellow) were grown in control (unstretched) and stretched devices. (b) Representative phase images of NAFs grown in control or stretched devices (after stretching), illustrating the random orientation of the cells attached to the PDMS membrane (scale bar is 50 μm). (c) Characteristic images demonstrating the immunostaining of fibronectin produced by unstretched NAFs, stretched NAFs, as well as CAFs (scale bar is 50 μm). (d) Average angles between fibronectin fibers measured as reported56 to compare fibronectin alignment. Both CAFs and stretched NAFs produced fibronectin with smaller angles than the fibronectin produced by unstretched NAFs. Error bars represent S.E.M for 50 cells from three individual experiments (*p < 0.002).

Mentions: To observe the effects of mechanical force in the activation of fibroblasts, we first examined the fibronectin fibers produced by human prostatic NAFs. We employed the microfluidic stretcher device to exert mechanical force on NAFs by loading the cells onto the suspended membrane and then stretching it (Fig. 3a). NAFs cultured on the PDMS membrane were stretched overnight under a consistent strain of ~4%, after which the stress was released and the NAFs were incubated for another ~24 hours before immunofluorescence staining was performed. Incubation for an additional ~24 hours was necessary to allow the fibroblasts sufficient time to deposit and assemble enough fibronectin for clear imaging. Note that even though the extra 24 hour incubation period was performed after the stretch was released, the fibronectin organization discussed below was still observable immediately after releasing the stretch, indicating that it was the stretching mechanical stimulus that initiated the phenotypic change, not the removal of the strain.


Stretching fibroblasts remodels fibronectin and alters cancer cell migration.

Ao M, Brewer BM, Yang L, Franco Coronel OE, Hayward SW, Webb DJ, Li D - Sci Rep (2015)

Stretched NAFs deposit fibronectin in an organized format.(a) NAFs (yellow) were grown in control (unstretched) and stretched devices. (b) Representative phase images of NAFs grown in control or stretched devices (after stretching), illustrating the random orientation of the cells attached to the PDMS membrane (scale bar is 50 μm). (c) Characteristic images demonstrating the immunostaining of fibronectin produced by unstretched NAFs, stretched NAFs, as well as CAFs (scale bar is 50 μm). (d) Average angles between fibronectin fibers measured as reported56 to compare fibronectin alignment. Both CAFs and stretched NAFs produced fibronectin with smaller angles than the fibronectin produced by unstretched NAFs. Error bars represent S.E.M for 50 cells from three individual experiments (*p < 0.002).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4321168&req=5

f3: Stretched NAFs deposit fibronectin in an organized format.(a) NAFs (yellow) were grown in control (unstretched) and stretched devices. (b) Representative phase images of NAFs grown in control or stretched devices (after stretching), illustrating the random orientation of the cells attached to the PDMS membrane (scale bar is 50 μm). (c) Characteristic images demonstrating the immunostaining of fibronectin produced by unstretched NAFs, stretched NAFs, as well as CAFs (scale bar is 50 μm). (d) Average angles between fibronectin fibers measured as reported56 to compare fibronectin alignment. Both CAFs and stretched NAFs produced fibronectin with smaller angles than the fibronectin produced by unstretched NAFs. Error bars represent S.E.M for 50 cells from three individual experiments (*p < 0.002).
Mentions: To observe the effects of mechanical force in the activation of fibroblasts, we first examined the fibronectin fibers produced by human prostatic NAFs. We employed the microfluidic stretcher device to exert mechanical force on NAFs by loading the cells onto the suspended membrane and then stretching it (Fig. 3a). NAFs cultured on the PDMS membrane were stretched overnight under a consistent strain of ~4%, after which the stress was released and the NAFs were incubated for another ~24 hours before immunofluorescence staining was performed. Incubation for an additional ~24 hours was necessary to allow the fibroblasts sufficient time to deposit and assemble enough fibronectin for clear imaging. Note that even though the extra 24 hour incubation period was performed after the stretch was released, the fibronectin organization discussed below was still observable immediately after releasing the stretch, indicating that it was the stretching mechanical stimulus that initiated the phenotypic change, not the removal of the strain.

Bottom Line: Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors.Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin.A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Vanderbilt University, Nashville, TN.

ABSTRACT
Most investigations of cancer-stroma interactions have focused on biochemical signaling effects, with much less attention being paid to biophysical factors. In this study, we investigated the role of mechanical stimuli on human prostatic fibroblasts using a microfluidic platform that was adapted for our experiments and further developed for both repeatable performance among multiple assays and for compatibility with high-resolution confocal microscopy. Results show that mechanical stretching of normal tissue-associated fibroblasts (NAFs) alters the structure of secreted fibronectin. Specifically, unstretched NAFs deposit and assemble fibronectin in a random, mesh-like arrangement, while stretched NAFs produce matrix with a more organized, linearly aligned structure. Moreover, the stretched NAFs exhibited an enhanced capability for directing co-cultured cancer cell migration in a persistent manner. Furthermore, we show that stretching NAFs triggers complex biochemical signaling events through the observation of increased expression of platelet derived growth factor receptor α (PDGFRα). A comparison of these behaviors with those of cancer-associated fibroblasts (CAFs) indicates that the observed phenotypes of stretched NAFs are similar to those associated with CAFs, suggesting that mechanical stress is a critical factor in NAF activation and CAF genesis.

No MeSH data available.


Related in: MedlinePlus