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Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression.

Qazi H, Shi ZD, Tarbell JM - PLoS ONE (2011)

Bottom Line: This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression.These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, City College of New York, City University of New York, New York, New York, United States of America.

ABSTRACT

Background: Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/principal findings: A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/significance: Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

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Related in: MedlinePlus

Darcy Flow Experimental Apparatus for the application of shear stress in 3D and Modified Boyden chamber invasion assay.(A) The flow apparatus applied a constant hydrostatic pressure via a double reservoir system composed of a larger reservoir [A] feeding flow media into the smaller syringe reservoir [B] and a pressure release tube [C], both of which were fastened within a rubber seal [D] to the cell culture insert [E]. The pressure release tube [C] ensured that there were no fluctuations in pressure applied across the collagen/cell suspension [F]. Hydrostatic pressure [ΔP] drove media throughout the thickness of the gel [L] and exerted shear stress on the cell membranes. Flow media filtrate was collected in another reservoir [G]. (B) At the end of the flow period, the inserts containing the cell suspensions [E] were decoupled from the apparatus and the invasion assay ensued. During the migration period, 10 nM TGF-α directed cells to migrate through 8 µm pores towards the underside of the filter. At the end of the migration period, cells on the underside of the inserts were stained and migration rates were quantified.
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pone-0020348-g001: Darcy Flow Experimental Apparatus for the application of shear stress in 3D and Modified Boyden chamber invasion assay.(A) The flow apparatus applied a constant hydrostatic pressure via a double reservoir system composed of a larger reservoir [A] feeding flow media into the smaller syringe reservoir [B] and a pressure release tube [C], both of which were fastened within a rubber seal [D] to the cell culture insert [E]. The pressure release tube [C] ensured that there were no fluctuations in pressure applied across the collagen/cell suspension [F]. Hydrostatic pressure [ΔP] drove media throughout the thickness of the gel [L] and exerted shear stress on the cell membranes. Flow media filtrate was collected in another reservoir [G]. (B) At the end of the flow period, the inserts containing the cell suspensions [E] were decoupled from the apparatus and the invasion assay ensued. During the migration period, 10 nM TGF-α directed cells to migrate through 8 µm pores towards the underside of the filter. At the end of the migration period, cells on the underside of the inserts were stained and migration rates were quantified.

Mentions: The boundaries between brain tumors and parenchyma have been shown to contain interstitial collagen type I among other matrix components, and some implanted glioma cell lines form interstitial ECM that consists primarily of interstitial collagen [20], [23], [30]. A model was developed in which glioma cells suspended in type I collagen were exposed to 3D fluid shear stress via the Darcy Flow Experimental Apparatus (Fig. 1A). High concentration Rat Tail Collagen Type I (BD Biosciences) was utilized as the stock and all dilutions were carried out using serum-containing media (DMEM containing 10% FBS; culture media). The walls of 12-well cell culture inserts (containing 8.0 µm pore filters [BD Falcon]) were pre-coated with 50 µl of 1 mg/ml collagen to prevent gel detachment or contraction by the suspended cells. 50,000 cells were suspended in 400 µl of 2 mg/ml collagen and immediately incubated within the pre-coated culture inserts for proper gelation to reduce settling of cells at the bottom due to gravity. The cell/collagen suspensions were incubated for 12 hours with 800 µl of serum-containing media in each well to allow sufficient time for cell spreading.


Fluid shear stress regulates the invasive potential of glioma cells via modulation of migratory activity and matrix metalloproteinase expression.

Qazi H, Shi ZD, Tarbell JM - PLoS ONE (2011)

Darcy Flow Experimental Apparatus for the application of shear stress in 3D and Modified Boyden chamber invasion assay.(A) The flow apparatus applied a constant hydrostatic pressure via a double reservoir system composed of a larger reservoir [A] feeding flow media into the smaller syringe reservoir [B] and a pressure release tube [C], both of which were fastened within a rubber seal [D] to the cell culture insert [E]. The pressure release tube [C] ensured that there were no fluctuations in pressure applied across the collagen/cell suspension [F]. Hydrostatic pressure [ΔP] drove media throughout the thickness of the gel [L] and exerted shear stress on the cell membranes. Flow media filtrate was collected in another reservoir [G]. (B) At the end of the flow period, the inserts containing the cell suspensions [E] were decoupled from the apparatus and the invasion assay ensued. During the migration period, 10 nM TGF-α directed cells to migrate through 8 µm pores towards the underside of the filter. At the end of the migration period, cells on the underside of the inserts were stained and migration rates were quantified.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3102715&req=5

pone-0020348-g001: Darcy Flow Experimental Apparatus for the application of shear stress in 3D and Modified Boyden chamber invasion assay.(A) The flow apparatus applied a constant hydrostatic pressure via a double reservoir system composed of a larger reservoir [A] feeding flow media into the smaller syringe reservoir [B] and a pressure release tube [C], both of which were fastened within a rubber seal [D] to the cell culture insert [E]. The pressure release tube [C] ensured that there were no fluctuations in pressure applied across the collagen/cell suspension [F]. Hydrostatic pressure [ΔP] drove media throughout the thickness of the gel [L] and exerted shear stress on the cell membranes. Flow media filtrate was collected in another reservoir [G]. (B) At the end of the flow period, the inserts containing the cell suspensions [E] were decoupled from the apparatus and the invasion assay ensued. During the migration period, 10 nM TGF-α directed cells to migrate through 8 µm pores towards the underside of the filter. At the end of the migration period, cells on the underside of the inserts were stained and migration rates were quantified.
Mentions: The boundaries between brain tumors and parenchyma have been shown to contain interstitial collagen type I among other matrix components, and some implanted glioma cell lines form interstitial ECM that consists primarily of interstitial collagen [20], [23], [30]. A model was developed in which glioma cells suspended in type I collagen were exposed to 3D fluid shear stress via the Darcy Flow Experimental Apparatus (Fig. 1A). High concentration Rat Tail Collagen Type I (BD Biosciences) was utilized as the stock and all dilutions were carried out using serum-containing media (DMEM containing 10% FBS; culture media). The walls of 12-well cell culture inserts (containing 8.0 µm pore filters [BD Falcon]) were pre-coated with 50 µl of 1 mg/ml collagen to prevent gel detachment or contraction by the suspended cells. 50,000 cells were suspended in 400 µl of 2 mg/ml collagen and immediately incubated within the pre-coated culture inserts for proper gelation to reduce settling of cells at the bottom due to gravity. The cell/collagen suspensions were incubated for 12 hours with 800 µl of serum-containing media in each well to allow sufficient time for cell spreading.

Bottom Line: This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression.These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, City College of New York, City University of New York, New York, New York, United States of America.

ABSTRACT

Background: Glioma cells are exposed to elevated interstitial fluid flow during the onset of angiogenesis, at the tumor periphery while invading normal parenchyma, within white matter tracts, and during vascular normalization therapy. Glioma cell lines that have been exposed to fluid flow forces in vivo have much lower invasive potentials than in vitro cell motility assays without flow would indicate.

Methodology/principal findings: A 3D Modified Boyden chamber (Darcy flow through collagen/cell suspension) model was designed to mimic the fluid dynamic microenvironment to study the effects of fluid shear stress on the migratory activity of glioma cells. Novel methods for gel compaction and isolation of chemotactic migration from flow stimulation were utilized for three glioma cell lines: U87, CNS-1, and U251. All physiologic levels of fluid shear stress suppressed the migratory activity of U87 and CNS-1 cell lines. U251 motility remained unaltered within the 3D interstitial flow model. Matrix Metalloproteinase (MMP) inhibition experiments and assays demonstrated that the glioma cells depended on MMP activity to invade, and suppression in motility correlated with downregulation of MMP-1 and MMP-2 levels. This was confirmed by RT-PCR and with the aid of MMP-1 and MMP-2 shRNA constructs.

Conclusions/significance: Fluid shear stress in the tumor microenvironment may explain reduced glioma invasion through modulation of cell motility and MMP levels. The flow-induced migration trends were consistent with reported invasive potentials of implanted gliomas. The models developed for this study imply that flow-modulated motility involves mechanotransduction of fluid shear stress affecting MMP activation and expression. These models should be useful for the continued study of interstitial flow effects on processes that affect tumor progression.

Show MeSH
Related in: MedlinePlus