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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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Silencing of Cofilin prevents mechanically stimulated invasion.A) Western blot of cofilin from lysates of HT1080 cells treated with off-target control siRNA (lane 1, from left), and cells cultured for 24 and 48 hours after nucleofection with cofilin siRNA (lanes 2 and 3 respectively). Cofilin expression is reduced 48 hours post-nucleofection. GAPDH was used as loading control. B) HT1080 cells nucleofected with control siRNA or Cofilin siRNA and cultured for 48 hours were seeded onto collagen/fibronectin matrices containing paramagnetic beads. The cells were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Invasion assays using control siRNA treated cells were repeated twice (15 fields were counted per trial). Stimulated cells had 3-fold higher invasion as compared to non-stimulated cells (P>0.05). The assay using cells when cofilin was silenced, was repeated four times (15 fields were counted per trial). The percent invasion between stimulated or non-stimulated cultures was insignificant (P>0.05). C) HT1080 cells were seeded onto collagen/fibronectin matrices containing paramagnetic beads. Cells treated with 2 µM Cytochalasin B or DMSO were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Data represents three independent assays.
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pone-0017277-g005: Silencing of Cofilin prevents mechanically stimulated invasion.A) Western blot of cofilin from lysates of HT1080 cells treated with off-target control siRNA (lane 1, from left), and cells cultured for 24 and 48 hours after nucleofection with cofilin siRNA (lanes 2 and 3 respectively). Cofilin expression is reduced 48 hours post-nucleofection. GAPDH was used as loading control. B) HT1080 cells nucleofected with control siRNA or Cofilin siRNA and cultured for 48 hours were seeded onto collagen/fibronectin matrices containing paramagnetic beads. The cells were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Invasion assays using control siRNA treated cells were repeated twice (15 fields were counted per trial). Stimulated cells had 3-fold higher invasion as compared to non-stimulated cells (P>0.05). The assay using cells when cofilin was silenced, was repeated four times (15 fields were counted per trial). The percent invasion between stimulated or non-stimulated cultures was insignificant (P>0.05). C) HT1080 cells were seeded onto collagen/fibronectin matrices containing paramagnetic beads. Cells treated with 2 µM Cytochalasin B or DMSO were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Data represents three independent assays.

Mentions: A functioning actin cytoskeleton is required for the invasiveness of a number of tumor cells [38], [39]. To confirm the significance of actin dynamics in HT1080 invasion into type I collagen/fibronectin matrix, Cytochalasin B or control DMSO treated cells were tested in the invasion assay. As anticipated, both the mechanically stimulated and the non-stimulated invasion were inhibited. Less than 1% of the cells treated with Cytochalasin B invaded irrespective of whether they were mechanically stimulated (Figure 5C). In comparison, 12% of non-stimulated and 29% of stimulated, DMSO treated control cells invaded into the matrix (Figure 5C). As expected, invasion into a 3D matrix is dependent on the dynamics of the actin cytoskeleton.


Cancer cell invasion is enhanced by applied mechanical stimulation.

Menon S, Beningo KA - PLoS ONE (2011)

Silencing of Cofilin prevents mechanically stimulated invasion.A) Western blot of cofilin from lysates of HT1080 cells treated with off-target control siRNA (lane 1, from left), and cells cultured for 24 and 48 hours after nucleofection with cofilin siRNA (lanes 2 and 3 respectively). Cofilin expression is reduced 48 hours post-nucleofection. GAPDH was used as loading control. B) HT1080 cells nucleofected with control siRNA or Cofilin siRNA and cultured for 48 hours were seeded onto collagen/fibronectin matrices containing paramagnetic beads. The cells were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Invasion assays using control siRNA treated cells were repeated twice (15 fields were counted per trial). Stimulated cells had 3-fold higher invasion as compared to non-stimulated cells (P>0.05). The assay using cells when cofilin was silenced, was repeated four times (15 fields were counted per trial). The percent invasion between stimulated or non-stimulated cultures was insignificant (P>0.05). C) HT1080 cells were seeded onto collagen/fibronectin matrices containing paramagnetic beads. Cells treated with 2 µM Cytochalasin B or DMSO were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Data represents three independent assays.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3040771&req=5

pone-0017277-g005: Silencing of Cofilin prevents mechanically stimulated invasion.A) Western blot of cofilin from lysates of HT1080 cells treated with off-target control siRNA (lane 1, from left), and cells cultured for 24 and 48 hours after nucleofection with cofilin siRNA (lanes 2 and 3 respectively). Cofilin expression is reduced 48 hours post-nucleofection. GAPDH was used as loading control. B) HT1080 cells nucleofected with control siRNA or Cofilin siRNA and cultured for 48 hours were seeded onto collagen/fibronectin matrices containing paramagnetic beads. The cells were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Invasion assays using control siRNA treated cells were repeated twice (15 fields were counted per trial). Stimulated cells had 3-fold higher invasion as compared to non-stimulated cells (P>0.05). The assay using cells when cofilin was silenced, was repeated four times (15 fields were counted per trial). The percent invasion between stimulated or non-stimulated cultures was insignificant (P>0.05). C) HT1080 cells were seeded onto collagen/fibronectin matrices containing paramagnetic beads. Cells treated with 2 µM Cytochalasin B or DMSO were cultured with or without stimulation for 48 hours and the percent of invading cells was calculated. Data represents three independent assays.
Mentions: A functioning actin cytoskeleton is required for the invasiveness of a number of tumor cells [38], [39]. To confirm the significance of actin dynamics in HT1080 invasion into type I collagen/fibronectin matrix, Cytochalasin B or control DMSO treated cells were tested in the invasion assay. As anticipated, both the mechanically stimulated and the non-stimulated invasion were inhibited. Less than 1% of the cells treated with Cytochalasin B invaded irrespective of whether they were mechanically stimulated (Figure 5C). In comparison, 12% of non-stimulated and 29% of stimulated, DMSO treated control cells invaded into the matrix (Figure 5C). As expected, invasion into a 3D matrix is dependent on the dynamics of the actin cytoskeleton.

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