Abstract
Cell invasion through the extracellular matrix (ECM) of connective tissue is an important biomechanical process, which plays a prominent role in tumor progression. The malignancy of tumors depends mainly on the capacity of cancer cells to migrate and metastasize. A prerequisite for metastasis is the invasion of cancer cells through connective tissue to targeted organs. Cellular stiffness and cytoskeletal remodeling dynamics have been proposed to affect the invasiveness of cancer cells. Here, this study investigated whether highly invasive cancer cells are capable of invading into dense 3D-ECMs with an average pore-size of 1.3 or 3.0 μm when phagocytized beads (2.7 and 4.5 μm diameter) increased their cellular stiffness and reduced their cytoskeletal remodeling dynamics compared to weakly invasive cancer cells. The phagocytized beads decreased the invasiveness of the α5β1high cancer cells into 3D-ECMs, whereas the invasiveness of the α5β1low cancer cells was not affected. The effect of phagocytized beads on the highly invasive α5β1high cells was abolished by specific knock-down of the α5 integrin subunit or addition of an anti-α5 integrin blocking antibody. Furthermore, the reduction of contractile forces using MLCK and ROCK inhibitors abolished the effect of phagocytized beads on the invasiveness of α5β1high cells. In addition, the cellular stiffness of α5β1high cells was increased after bead phagocytosis, whereas the bead phagocytosis did not alter the stiffness of α5β1low cells. Taken together, the α5β1 integrin dependent invasiveness was reduced after bead phagocytosis by altered biomechanical properties, suggesting that the α5β1high cells need an appropriate intermediate cellular stiffness to overcome the steric hindrance of 3D-ECMs, whereas the α5β1low cells were not affected by phagocytized beads.
Similar content being viewed by others
Abbreviations
- ECM:
-
Extra cellular matrix
- FN:
-
Fibronectin
- MLCK:
-
Myosin light chain kinase
- COL:
-
Collagen
References
Batlle, E., Sancho, E., Franci, C., Dominguez, D., Monfar, M., Baulida, J., et al. (2000). The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells. Nature Cell Biology, 2, 84–89.
Behrens, J., Mareel, M. M., Van Roy, F. M., & Birchmeier, W. (1989). Dissecting tumor cell invasion: Epithelial cells acquire invasive properties after the loss of uvomorulin-mediated cell–cell adhesion. Journal of Cell Biology, 108, 2435–2447.
Cano, A., Perez-Moreno, M. A., Rodrigo, I., Locascio, A., Blanco, M. J., del Barrio, M. G., et al. (2000). The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression. Nature Cell Biology, 2, 76–83.
Danen, E. H., van Rheenen, J., Franken, W., Huveneers, S., Sonneveld, P., Jalink, K., et al. (2005). Integrins control motile strategy through a Rho-cofilin pathway. Journal of Cell Biology, 169, 515–526.
De Craene, B., Gilbert, B., Stove, C., Bruyneel, E., van Roy, F., & Berx, G. (2005). The transcription factor snail induces tumor cell invasion through modulation of the epithelial cell differentiation program. Cancer Research, 65, 6237–6244.
Frixen, U. H., Behrens, J., Sachs, M., Eberle, G., Voss, B., & Warda, A. (1991). E-cadherin-mediated cell–cell adhesion prevents invasiveness of human carcinoma cells. Journal of Cell Biology, 113, 173–185.
Vleminckx, K., Vakaet, L, Jr, Mareel, M., Fiers, W., & van Roy, F. (1991). Genetic manipulation of E-cadherin expression by epithelial tumor cells reveals an invasion suppressor role. Cell, 66, 107–119.
Al-Mehdi, A. B., Tozawa, K., Fisher, A. B., Shientag, L., Lee, A., & Muschel, R. J. (2000). Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: A new model for metastasis. Nature Medicine, 6, 100–102.
Steeg, P. S. (2006). Tumor metastasis: Mechanistic insights and clinical challenges. Nature Medicine, 12, 895–904.
Buckley, C. D., Doyonnas, R., Newton, J. P., Blystone, S. D., Brown, E. J., Watt, S. M., et al. (1996). Identification of alpha v beta 3 as a heterotypic ligand for CD31/PECAM-1. Journal of Cell Science, 109, 437–445.
Leroy-Dudal, J., Demeilliers, C., Gallet, O., Pauthe, E., Dutoit, S., Agniel, R., et al. (2005). Transmigration of human ovarian adenocarcinoma cells through endothelial extracellular matrix involves alphav integrins and the participation of MMP2. International Journal of Cancer, 114, 531–543.
Voura, E. B., Ramjeesingh, R. A., Montgomery, A. M., & Siu, C. H. (2001). Involvement of integrin alpha(v)beta(3) and cell adhesion molecule L1 in transendothelial migration of melanoma cells. Molecular Biology of the Cell, 12, 2699–2710.
Playford, M. P., & Schaller, M. D. (2004). The interplay between Src and integrins in normal and tumor biology. Oncogene, 23, 7928–7946.
Burridge, K., & Wennerberg, K. (2004). Rho and Rac take center stage. Cell, 116, 167–179.
Bauer, K., Mierke, C., & Behrens, J. (2007). Expression profiling reveals genes associated with transendothelial migration of tumor cells: A functional role for alpha v beta3 integrin. International Journal of Cancer, 121, 1910–1918.
Mierke, C. T., Frey, B., Fellner, M., Herrmann, M., & Fabry, B. (2011). Integrin α5β1 facilitates cancer cell invasion through enhanced contractile forces. Journal of Cell Science, 124, 369–383.
Guck, J., Schinkinger, S., Lincoln, B., Wottawah, F., Ebert, S., Romeyke, M., et al. (2005). Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophysical Journal, 88, 3689–3698.
Suresh, S. (2007). Biomechanics and biophysics of cancer cells. Acta Biomaterialia, 3, 413–438.
Hynes, R. O. (2002). Integrins: Bidirectional, allosteric signaling machines. Cell, 110, 673–687.
Arnaout, M. A., Goodman, S. L., & Xiong, J. P. (2007). Structure and mechanics of integrin-based cell adhesion. Current Opinion in Cell Biology, 19, 495–507.
Damsky, C. H., Knudsen, K. A., Bradley, D., Buck, C. A., & Horwitz, A. F. (1985). Distribution of the cell substratum attachment (CSAT) antigen on myogenic and fibroblastic cells in culture. Journal of Cell Biology, 100, 1528–1539.
Moser, M., Legate, K. R., Zent, R., & Fässler, R. (2009). The tail of integrins, talin, and kindlins. Science, 324, 895–899.
Geiger, B., Bershadsky, A., Pankov, R., & Yamada, K. M. (2001). Transmembrane crosstalk between the extracellular matrix–cytoskeleton crosstalk. Nature Reviews Molecular Cell Biology, 2, 793–805.
Geiger, B., Yamada, KM. (2011). Molecular architecture and function of matrix adhesions. Cold Spring Harbor Perspectives in Biology , 3 (5). doi: 10.1101/cshperspect.a005033.
Balaban, N. Q., Schwarz, U. S., Riveline, D., Goichberg, P., Tzur, G., Sabanay, I., et al. (2001). Force and focal adhesion assembly: A close relationship studied using elastic micropatterned substrates. Nature Cell Biology, 3, 466–472.
Loftus, J. C., & Liddington, R. C. (1997). Cell adhesion in vascular biology. New insights into integrin-ligand interaction. The Journal of Clinical Investigation, 99, 2302–2306.
Palecek, S. P., Loftus, J. C., Ginsberg, M. H., Lauffenburger, D. A., & Horwitz, A. F. (1997). Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature, 385, 537–540.
Zaman, M. H., Trapani, L. M., Siemeski, A., Mackellar, D., Gong, H., Kamm, R. D., et al. (2006). Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis. Proceedings of the National Academy of Sciences of the United States of America, 103, 10889–10894.
Elson, E. L. (1988). Cellular mechanics as an indicator of cytoskeletal structure and function. Annual Review of Biophysics and Biophysical Chemistry, 17, 397–430.
Giannone, G., & Sheetz, M. P. (2006). Substrate rigidity and force define form through tyrosine phosphatase and kinase pathways. Trends in Cell Biology, 16, 213–223.
Friedland, J. C., Lee, M. H., & Boettiger, D. (2009). Mechanically activated integrin switch controls alpha5beta1 function. Science, 323, 642–644.
Gallant, N. D., Michael, K. E., & Garcia, A. J. (2005). Cell adhesion strengthening: Contributions of adhesive area, integrin binding, and focal adhesion assembly. Molecular Biology of the Cell, 16, 4329–4340.
Paszek, M. J., Zahir, N., Johnson, K. R., Lakins, J. N., Rozenberg, G. I., Gefen, A., et al. (2005). Tensional homeostasis and the malignant phenotype. Cancer Cell, 8, 241–254.
Mierke, C. T., Kollmannsberger, P., Paranhos-Zitterbart, D., Smith, J., Fabry, B., & Goldmann, W. H. (2008). Mechano-coupling and regulation of contractility by the vinculin tail domain. Biophysical Journal, 94, 661–670.
Mierke, C. T., Kollmannsberger, P., Zitterbart, D. P., Diez, G., Koch, T. M., Marg, S., et al. (2010). Vinculin facilitates cell invasion into three-dimensional collagen matrices. Journal of Biological Chemistry, 285, 13121–13130.
Caswell, P. T., Chan, M., Lindsay, A. J., McCaffrey, M. W., Boettiger, D., & Norman, J. C. (2008). Rab-coupling protein coordinates recycling of alpha5beta1 integrin and EGFR1 to promote cell migration in 3D microenvironments. Journal of Cell Biology, 183, 143–155.
Caswell, P. T., Spence, H. J., Parsons, M., White, D. P., Clark, K., Cheng, K., et al. (2007). Rab25 associates with alpha5beta1 integrin to promote invasive migration in 3D microenvironments. Developmental Cell, 13, 496–510.
Qian, F., Zhang, Z. C., Wu, X. F., Li, Y. P., & Xu, Q. (2005). Interaction between integrin alpha(5) and fibronectin is required for metastasis of B16F10 melanoma cells. Biochemical and Biophysical Research Communications, 333, 1269–1275.
Sawada, K., Mitra, A. K., Radjabi, A. R., Bhaskar, V., Kistner, E. O., Tretiakova, M., et al. (2008). Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target. Cancer Research, 68, 2329–2339.
Wu, H., Liang, Y. L., Li, Z., Jin, J., Zhang, W., Duan, L., et al. (2006). Positive expression of E-cadherin suppresses cell adhesion to fibronectin via reduction of alpha5beta1 integrin in human breast carcinoma cells. Journal of Cancer Research and Clinical Oncology, 132, 795–803.
Schirner, M., Herzberg, F., Schmidt, R., Streit, M., Schoning, M., Hummel, M., et al. (1998). Integrin alpha5beta1: A potent inhibitor of experimental lung metastasis. Clinical and Experimental Metastasis, 16, 427–435.
Tani, N., Higashiyama, S., Kawaguchi, N., Madarame, J., Ota, I., Ito, Y., et al. (2003). Expression level of integrin alpha 5 on tumour cells affects the rate of metastasis to the kidney. British Journal of Cancer, 88, 327–333.
Taverna, D., Ullman-Cullere, M., Rayburn, H., Bronson, R. T., & Hynes, R. O. (1998). A test of the role of alpha5 integrin/fibronectin interactions in tumorigenesis. Cancer Research, 58, 848–853.
Friedl, P., & Brocker, E. B. (2000). The biology of cell locomotion within three-dimensional extracellular matrix. Cellular and Molecular Life Sciences, 57, 41–64.
Mierke, C. T., Zitterbart, D. P., Kollmannsberger, P., Raupach, C., Schlotzer-Schrehardt, U., Goecke, T., et al. (2008). Breakdown of the endothelial barrier function in tumor cell transmigration. Biophysical Journal, 94, 2832–2846.
Webb, D. J., Donais, K., Whitmore, L. A., Thomas, S. M., Turner, C. E., Parsons, J. T., et al. (2004). FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nature Cell Biology, 6, 154–161.
Mierke, C. T., Bretz, N., & Altevogt, P. (2011). Contractile forces contribute to increased GPI-anchored receptor CD24 facilitated cancer cell invasion. The Journal of Biological Chemistry, 286, 34858–34871.
Wolf, K., Mazo, I., Leung, H., Engelke, K., von Andrian, U. H., Deryugina, E. I., et al. (2003). Compensation mechanism in tumor cell migration: Mesenchymal-amoeboid transition after blocking of pericellular proteolysis. Journal of Cell Biology, 160, 267–277.
Wang, N., Butler, J. P., & Ingber, D. E. (1993). Mechanotransduction across the cell surface and through the cytoskeleton. Science, 260, 1124–1127.
Fabry, B., Maksym, G. N., Shore, S. A., Moore, P. E., Panettieri, R. A, Jr, Butler, J. P., et al. (2001). Time course and heterogeneity of contractile responses in cultured human airway smooth muscle cells. Journal of Applied Physiology, 91, 986–994.
Chen, J., Fabry, B., Schiffrin, E. L., & Wang, N. (2001). Twisting integrin receptors increases endothelin-1 gene expression in endothelial cells. American Journal of Physiology-Cell Physiology, 280, C1475–C1484.
Mickel, W., Muenster, S., Jawerth, L. M., Vader, D. A., Weitz, D. A., Sheppard, A. P., et al. (2008). Robust pore size analysis of filamentous networks from 3d confocal microscopy. Biophysical Journal, 95, 6072–6080.
Fritsch, A., Höckel, M., Kiessling, T., Nnetu, K. D., Wetzel, F., Zink, M., et al. (2010). Are biomechanical changes necessary for tumour progression? Nature Physics, 6, 730–732.
Brábek, J., Mierke, C. T., Rösel, D., Veselý, P., & Fabry, B. (2010). The role of the tissue microenvironment in the regulation of cancer cell motility and invasion. Cell Communication and Signaling, 22, 1–8.
Kumar, S., & Weaver, V. M. (2009). Mechanics, malignancy, and metastasis: The force journey of a tumor cell. Cancer and Metastasis Reviews, 28, 113–127.
Mierke, C. T. (2011). The biomechanical properties of 3d extracellular matrices and embedded cells regulate the invasiveness of cancer cells. Cell Biochemistry and Biophysics, 61, 217–236.
Wei, Y., Czekay, R. P., Robillard, L., Kugler, M. C., Zhang, F., Kim, K. K., et al. (2005). Regulation of alpha5beta1 integrin conformation and function by urokinase receptor binding. Journal of Cell Biology, 168, 501–511.
Huveneers, S., Truong, H., Fassler, R., Sonnenberg, A., & Danen, E. H. (2008). Binding of soluble fibronectin to integrin alpha5 beta1-link to focal adhesion redistribution and contractile shape. Journal of Cell Science, 121, 2452–2462.
Mierke, C. T. (2011). Cancer cells regulate biomechanical properties of human microvascular endothelial cells. Journal of Biological Chemistry, 286, 40025–40037.
Lu, H., Mabilat, C., Yeh, P., Guitton, J. D., Li, H., Pouchelet, M., et al. (1996). Blockage of urokinase receptor reduces in vitro the motility and the deformability of endothelial cells. FEBS Letters, 380, 21–24.
Arpaia, E., Blaser, H., Quintela-Fandino, M., Duncan, G., Leong, H. S., Ablack, A., et al. (2012). The interaction between caveolin-1 and Rho-GTPases promotes metastasis by controlling the expression of alpha5-integrin and the activation of Src, Ras and Erk. Oncogene, 31, 884–896.
Raupach, C., Zitterbart, D. P., Mierke, C. T., Metzner, C., Müller, F. A., & Fabry, B. (2007). Stress fluctuations and motion of cytoskeletal-bound markers. Physical Review E: Statistical, Nonlinear, and Soft Matter Physics, 76(1 Pt 1), 011918.
Dieterich, P., Klages, R., Preuss, R., & Schwab, A. (2008). Anomalous dynamics of cell migration. Proceedings of the National Academy of Sciences of the United States of America, 105, 459–463.
Oakes, P. W., Patel, D. C., Morin, N. A., Zitterbart, D. P., Fabry, B., Reichner, J. S., et al. (2009). Neutrophil morphology and migration are affected by substrate elasticity. Blood, 114, 1387–1395.
Potdar, A. A., Jeon, J., Weaver, A. M., Quaranta, V., & Cummings, P. T. (2010). Human mammary epithelial cells exhibit a bimodal correlated random walk pattern. PLoS ONE, 5, e9636.
Kollmannsberger, P., Mierke, C. T., & Fabry, B. (2011). Nonlinear viscoelasticity of adherent cells is controlled by cytoskeletal tension. Soft Matter, 7, 3127–3132.
Mijailovich, S. M., Kojic, M., Zivkovic, M., Fabry, B., & Fredberg, J. J. (2002). A finite element model of cell deformation during magnetic bead twisting. Journal of Applied Physiology, 93, 1429–1436.
Kasza, K. E., Nakamura, F., Hu, S., Kollmannsberger, P., Fabry, B., Stossel, T. P., et al. (2009). Filamin A is essential for active cell stiffening but not passive stiffening under external force. Biophysical Journal, 96, 4326–4335.
Fredberg, J. J., Jones, K. A., Nathan, M., Raboudi, S., Prakash, Y. S., Shore, S. A., et al. (1996). Friction in airway smooth muscle: Mechanism, latch, and implications in asthma. Journal of Applied Physiology, 81, 2703–2712.
Möller, W., Stahlhofen, W., & Roth, C. (1990). Improved spinning top aerosol generator for the production of high concentrated ferromagnetic aerosols. Journal of Aerosol Science, 21, S435–S438.
Eekhoff, A., Bonakdar, N., Alonso, J. L., Hoffmann, B., & Goldmann, W. H. (2011). Glomerular podocytes: A study of mechanical properties and mechano-chemical signaling. Biochemical and Biophysical Research Communications, 406, 229–233.
Acknowledgments
I thank Dieter Freitag for excellent help with the magnetic twisting cytometry, Ben Fabry and Wolfgang H. Goldmann for helpful discussions, and Barbara Reischl, Christine Albert and Werner Schneider for excellent technical assistance. This work was supported by the Deutsche Krebshilfe (109432).
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mierke, C.T. Phagocytized Beads Reduce the α5β1 Integrin Facilitated Invasiveness of Cancer Cells by Regulating Cellular Stiffness. Cell Biochem Biophys 66, 599–622 (2013). https://doi.org/10.1007/s12013-012-9506-3
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12013-012-9506-3