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Cancer cell invasion is enhanced by applied mechanical stimulation.

Menon S, Beningo KA - PLoS ONE (2011)

Bottom Line: We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments.Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion.We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.

ABSTRACT
Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.

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Enhanced invasion of mechanically stimulated cultures of cancer cells.A) HT1080 fibrosarcoma cells were seeded onto type I collagen/fibronectin matrices containing paramagnetic beads and cultured either under magnetic stimulation or without stimulation. A combined phase and fluorescent image of a mechanically stimulated culture were superimposed. The solid arrow points to a cell that has invaded. The dotted arrow indicates a second cell within another focal plane. The empty arrow points to a fluorescent paramagnetic bead. Mag. Bar  = 50 µm. B) Invasion of HT1080 cells under mechanically stimulated and non-stimulated conditions was performed in matrices containing either type I collagen (2.5 mg/ml) or both type I collagen and fibronectin, or collagen/fibronectin in the presence or absence of RGD peptide. 25 fields of cells were counted 24 hours after seeding at multiple depths within each matrix beginning 5 µm below the surface of the matrix and progressing towards the farthest depth of 800 µm. The percent of invading cells was 2-fold higher in stimulated cultures when compared to controls (P<0.05) in matrices containing both ECM proteins. Similar results were obtained when the control peptide GRGES was added to the media. The percent invasion was approximately the same with or without stimulation when fibronectin was absent. Addition of the GRGDS peptide also resulted in inhibition of enhanced invasion upon mechanical stimulation. C) A 20-fold difference in the frequency of stimulation does not influence the percent of cell invasion. The percent of invading cells 24 hours after stimulation at magnetic rotation speeds of 8, 90 and 160 rpm (0.13, 1.5 and 2.6 Hz). An insignificant difference was found between cells stimulated at 8 and 160 rpm (P>0.05). Data represents three independent experiments, of 25 fields. D) Type I collagen/fibronectin matrices containing paramagnetc beads were pre-stimulated for 24 hours. These matrices were then seeded with HT1080 cells and counted 24 hours after seeding, during which period both the pre-stimulated and the control plates were not stimulated (left panel). These cultures were then either continued or placed over the magnet (right panel), data obtained 24 hours after stimulation. Data represents two independent assays of 15 fields of cells at a depth range of 800 µm. Two-tailed analysis using student t-test.
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pone-0017277-g003: Enhanced invasion of mechanically stimulated cultures of cancer cells.A) HT1080 fibrosarcoma cells were seeded onto type I collagen/fibronectin matrices containing paramagnetic beads and cultured either under magnetic stimulation or without stimulation. A combined phase and fluorescent image of a mechanically stimulated culture were superimposed. The solid arrow points to a cell that has invaded. The dotted arrow indicates a second cell within another focal plane. The empty arrow points to a fluorescent paramagnetic bead. Mag. Bar  = 50 µm. B) Invasion of HT1080 cells under mechanically stimulated and non-stimulated conditions was performed in matrices containing either type I collagen (2.5 mg/ml) or both type I collagen and fibronectin, or collagen/fibronectin in the presence or absence of RGD peptide. 25 fields of cells were counted 24 hours after seeding at multiple depths within each matrix beginning 5 µm below the surface of the matrix and progressing towards the farthest depth of 800 µm. The percent of invading cells was 2-fold higher in stimulated cultures when compared to controls (P<0.05) in matrices containing both ECM proteins. Similar results were obtained when the control peptide GRGES was added to the media. The percent invasion was approximately the same with or without stimulation when fibronectin was absent. Addition of the GRGDS peptide also resulted in inhibition of enhanced invasion upon mechanical stimulation. C) A 20-fold difference in the frequency of stimulation does not influence the percent of cell invasion. The percent of invading cells 24 hours after stimulation at magnetic rotation speeds of 8, 90 and 160 rpm (0.13, 1.5 and 2.6 Hz). An insignificant difference was found between cells stimulated at 8 and 160 rpm (P>0.05). Data represents three independent experiments, of 25 fields. D) Type I collagen/fibronectin matrices containing paramagnetc beads were pre-stimulated for 24 hours. These matrices were then seeded with HT1080 cells and counted 24 hours after seeding, during which period both the pre-stimulated and the control plates were not stimulated (left panel). These cultures were then either continued or placed over the magnet (right panel), data obtained 24 hours after stimulation. Data represents two independent assays of 15 fields of cells at a depth range of 800 µm. Two-tailed analysis using student t-test.

Mentions: These cell types were tested individually for their ability to respond to the mechanical stimulation provided in the assay. In brief, cells were seeded onto prepared matrices, as described in methods, and allowed to adhere for 30 minutes before beginning the stimulation. Cells cultured on matrices of identical composition, but not subjected to magnetic stimulation, served as controls. Cells cultured on matrices lacking magnetic beads, but subjected to magnetic stimulation served as additional controls. Invasion was observed under the microscope beginning at 5 µm from the surface to a depth of 800 µm within the matrix (Figure 3A). The number of invading and non-invading cells were counted after 24 hours of stimulation and calculated as the percent invasion.


Cancer cell invasion is enhanced by applied mechanical stimulation.

Menon S, Beningo KA - PLoS ONE (2011)

Enhanced invasion of mechanically stimulated cultures of cancer cells.A) HT1080 fibrosarcoma cells were seeded onto type I collagen/fibronectin matrices containing paramagnetic beads and cultured either under magnetic stimulation or without stimulation. A combined phase and fluorescent image of a mechanically stimulated culture were superimposed. The solid arrow points to a cell that has invaded. The dotted arrow indicates a second cell within another focal plane. The empty arrow points to a fluorescent paramagnetic bead. Mag. Bar  = 50 µm. B) Invasion of HT1080 cells under mechanically stimulated and non-stimulated conditions was performed in matrices containing either type I collagen (2.5 mg/ml) or both type I collagen and fibronectin, or collagen/fibronectin in the presence or absence of RGD peptide. 25 fields of cells were counted 24 hours after seeding at multiple depths within each matrix beginning 5 µm below the surface of the matrix and progressing towards the farthest depth of 800 µm. The percent of invading cells was 2-fold higher in stimulated cultures when compared to controls (P<0.05) in matrices containing both ECM proteins. Similar results were obtained when the control peptide GRGES was added to the media. The percent invasion was approximately the same with or without stimulation when fibronectin was absent. Addition of the GRGDS peptide also resulted in inhibition of enhanced invasion upon mechanical stimulation. C) A 20-fold difference in the frequency of stimulation does not influence the percent of cell invasion. The percent of invading cells 24 hours after stimulation at magnetic rotation speeds of 8, 90 and 160 rpm (0.13, 1.5 and 2.6 Hz). An insignificant difference was found between cells stimulated at 8 and 160 rpm (P>0.05). Data represents three independent experiments, of 25 fields. D) Type I collagen/fibronectin matrices containing paramagnetc beads were pre-stimulated for 24 hours. These matrices were then seeded with HT1080 cells and counted 24 hours after seeding, during which period both the pre-stimulated and the control plates were not stimulated (left panel). These cultures were then either continued or placed over the magnet (right panel), data obtained 24 hours after stimulation. Data represents two independent assays of 15 fields of cells at a depth range of 800 µm. Two-tailed analysis using student t-test.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3040771&req=5

pone-0017277-g003: Enhanced invasion of mechanically stimulated cultures of cancer cells.A) HT1080 fibrosarcoma cells were seeded onto type I collagen/fibronectin matrices containing paramagnetic beads and cultured either under magnetic stimulation or without stimulation. A combined phase and fluorescent image of a mechanically stimulated culture were superimposed. The solid arrow points to a cell that has invaded. The dotted arrow indicates a second cell within another focal plane. The empty arrow points to a fluorescent paramagnetic bead. Mag. Bar  = 50 µm. B) Invasion of HT1080 cells under mechanically stimulated and non-stimulated conditions was performed in matrices containing either type I collagen (2.5 mg/ml) or both type I collagen and fibronectin, or collagen/fibronectin in the presence or absence of RGD peptide. 25 fields of cells were counted 24 hours after seeding at multiple depths within each matrix beginning 5 µm below the surface of the matrix and progressing towards the farthest depth of 800 µm. The percent of invading cells was 2-fold higher in stimulated cultures when compared to controls (P<0.05) in matrices containing both ECM proteins. Similar results were obtained when the control peptide GRGES was added to the media. The percent invasion was approximately the same with or without stimulation when fibronectin was absent. Addition of the GRGDS peptide also resulted in inhibition of enhanced invasion upon mechanical stimulation. C) A 20-fold difference in the frequency of stimulation does not influence the percent of cell invasion. The percent of invading cells 24 hours after stimulation at magnetic rotation speeds of 8, 90 and 160 rpm (0.13, 1.5 and 2.6 Hz). An insignificant difference was found between cells stimulated at 8 and 160 rpm (P>0.05). Data represents three independent experiments, of 25 fields. D) Type I collagen/fibronectin matrices containing paramagnetc beads were pre-stimulated for 24 hours. These matrices were then seeded with HT1080 cells and counted 24 hours after seeding, during which period both the pre-stimulated and the control plates were not stimulated (left panel). These cultures were then either continued or placed over the magnet (right panel), data obtained 24 hours after stimulation. Data represents two independent assays of 15 fields of cells at a depth range of 800 µm. Two-tailed analysis using student t-test.
Mentions: These cell types were tested individually for their ability to respond to the mechanical stimulation provided in the assay. In brief, cells were seeded onto prepared matrices, as described in methods, and allowed to adhere for 30 minutes before beginning the stimulation. Cells cultured on matrices of identical composition, but not subjected to magnetic stimulation, served as controls. Cells cultured on matrices lacking magnetic beads, but subjected to magnetic stimulation served as additional controls. Invasion was observed under the microscope beginning at 5 µm from the surface to a depth of 800 µm within the matrix (Figure 3A). The number of invading and non-invading cells were counted after 24 hours of stimulation and calculated as the percent invasion.

Bottom Line: We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments.Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion.We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Wayne State University, Detroit, Michigan, United States of America.

ABSTRACT
Metastatic cells migrate from the site of the primary tumor, through the stroma, into the blood and lymphatic vessels, finally colonizing various other tissues to form secondary tumors. Numerous studies have been done to identify the stimuli that drive the metastatic cascade. This has led to the identification of multiple biochemical signals that promote metastasis. However, information on the role of mechanical factors in cancer metastasis has been limited to the affect of compliance. Interestingly, the tumor microenvironment is rich in many cell types including highly contractile cells that are responsible for extensive remodeling and production of the dense extracellular matrix surrounding the cancerous tissue. We hypothesize that the mechanical forces produced by remodeling activities of cells in the tumor microenvironment contribute to the invasion efficiency of metastatic cells. We have discovered a significant difference in the extent of invasion in mechanically stimulated verses non-stimulated cell culture environments. Furthermore, this mechanically enhanced invasion is dependent upon substrate protein composition, and influenced by topography. Finally, we have found that the protein cofilin is needed to sense the mechanical stimuli that enhances invasion. We conclude that other types of mechanical signals in the tumor microenvironment, besides the rigidity, can enhance the invasive abilities of cancer cells in vitro. We further propose that in vivo, non-cancerous cells located within the tumor micro-environment may be capable of providing the necessary mechanical stimulus during the remodeling of the extracellular matrix surrounding the tumor.

Show MeSH
Related in: MedlinePlus