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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

Exposure to shearing forces did not induce cell apoptosis or necrosis.At the end of the migration period, both non-sheared cells (in control gels; A, C) and cells in gels exposed to 0.55 dynes/cm2 shear stress (B, D) were stained either by the Vybrant Apoptosis Assay Kit no. 2 or by Calcein AM. (A, B) There was no evidence of apoptosis being induced in the U87 cells as a result of exposure to the higher levels of shearing forces in this experiment; apoptotic cells were stained with Alexa Fluor 488 annexin V (green) and necrotic cells were stained with propidium iodide (red). (C, D) Calcein AM (green) staining indicates that a majority of the cells remained viable and cell morphology was normal in both gels containing non-sheared U87 cells and cells exposed to 0.55 dynes/cm2 shear stress for four hours.
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pone-0020348-g005: Exposure to shearing forces did not induce cell apoptosis or necrosis.At the end of the migration period, both non-sheared cells (in control gels; A, C) and cells in gels exposed to 0.55 dynes/cm2 shear stress (B, D) were stained either by the Vybrant Apoptosis Assay Kit no. 2 or by Calcein AM. (A, B) There was no evidence of apoptosis being induced in the U87 cells as a result of exposure to the higher levels of shearing forces in this experiment; apoptotic cells were stained with Alexa Fluor 488 annexin V (green) and necrotic cells were stained with propidium iodide (red). (C, D) Calcein AM (green) staining indicates that a majority of the cells remained viable and cell morphology was normal in both gels containing non-sheared U87 cells and cells exposed to 0.55 dynes/cm2 shear stress for four hours.

Mentions: Exposure to four hours of 0.55 dynes/cm2 shear stress did not appear to induce cell apoptosis or necrosis in the U87 cell line (Fig. 5A–B). Calcein AM staining indicated that most of the cells remained viable, morphologically normal, and well spread within both control and flow (sheared) suspensions (Fig. 5C–D). Similar results were observed with the CNS-1 cell line (Supplementary Fig. S5). The cells that were apoptotic or necrotic deviated from their normal spindle-like appearance to a more contractile morphology. It should be noted that a contractile morphology itself does not imply suppressed migration [38]. A majority of the apoptotic and necrotic cells were observed towards the top of the gel (gel/air interface).


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)

Exposure to shearing forces did not induce cell apoptosis or necrosis.At the end of the migration period, both non-sheared cells (in control gels; A, C) and cells in gels exposed to 0.55 dynes/cm2 shear stress (B, D) were stained either by the Vybrant Apoptosis Assay Kit no. 2 or by Calcein AM. (A, B) There was no evidence of apoptosis being induced in the U87 cells as a result of exposure to the higher levels of shearing forces in this experiment; apoptotic cells were stained with Alexa Fluor 488 annexin V (green) and necrotic cells were stained with propidium iodide (red). (C, D) Calcein AM (green) staining indicates that a majority of the cells remained viable and cell morphology was normal in both gels containing non-sheared U87 cells and cells exposed to 0.55 dynes/cm2 shear stress for four hours.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0020348-g005: Exposure to shearing forces did not induce cell apoptosis or necrosis.At the end of the migration period, both non-sheared cells (in control gels; A, C) and cells in gels exposed to 0.55 dynes/cm2 shear stress (B, D) were stained either by the Vybrant Apoptosis Assay Kit no. 2 or by Calcein AM. (A, B) There was no evidence of apoptosis being induced in the U87 cells as a result of exposure to the higher levels of shearing forces in this experiment; apoptotic cells were stained with Alexa Fluor 488 annexin V (green) and necrotic cells were stained with propidium iodide (red). (C, D) Calcein AM (green) staining indicates that a majority of the cells remained viable and cell morphology was normal in both gels containing non-sheared U87 cells and cells exposed to 0.55 dynes/cm2 shear stress for four hours.
Mentions: Exposure to four hours of 0.55 dynes/cm2 shear stress did not appear to induce cell apoptosis or necrosis in the U87 cell line (Fig. 5A–B). Calcein AM staining indicated that most of the cells remained viable, morphologically normal, and well spread within both control and flow (sheared) suspensions (Fig. 5C–D). Similar results were observed with the CNS-1 cell line (Supplementary Fig. S5). The cells that were apoptotic or necrotic deviated from their normal spindle-like appearance to a more contractile morphology. It should be noted that a contractile morphology itself does not imply suppressed migration [38]. A majority of the apoptotic and necrotic cells were observed towards the top of the gel (gel/air interface).

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