Abstract
In the continuous search for better tissue engineering scaffolds it has become increasingly clear that the substrate properties dramatically affect cell responses. Here we compared cells from a physiologically stiff tissue, melanoma, to cells isolated from a physiologically soft tissue, brain. We measured the cell line responses to laminin immobilized onto glass or polyacrylamide hydrogels tuned to have a Young’s modulus ranging from 1 to 390 kPa. Single cells were analyzed for spreading area, shape, total actin content, actin-based morphological features and modification of immobilized laminin. Both cell types exhibited stiffness- and laminin concentration-dependent responses on polyacrylamide and glass. Melanoma cells exhibited very little spreading and were rounded on soft (1, 5, and 15 kPa) hydrogels while cells on stiff (40, 100, and 390 kPa) hydrogels were spread and had a polarized cell shape with large lamellipodia. On rigid glass surfaces, spreading and actin-based morphological features were not observed until laminin concentration was much higher. Similarly, increased microglia cell spreading and presence of actin-based structures were observed on stiff hydrogels. However, responses on rigid glass surfaces were much different. Microglia cells had large spreading areas and elongated shapes on glass compared to hydrogels even when immobilized laminin density was consistent on all gels. While cell spreading and shape varied with Young’s modulus of the hydrogel, the concentration of f-actin was constant. A decrease in laminin immunofluorescence was associated with melanoma and microglia cell spreading on glass with high coating concentration of laminin, indicating modification of immobilized laminin triggered by supraphysiologic stiffness and high ligand density. These results suggest that some cell lines are more sensitive to mechanical properties matching their native tissue environment while other cell lines may require stiffness and extracellular ligand density well above physiologic tissue before saturation in cell spreading, elongation and cytoskeletal re-organization are reached.
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Engler, A. J., Sen, S., Sweeney, H. L., & Discher, D. E. (2006). Matrix elasticity directs stem cell lineage specification. Cell, 126, 677–689.
Tilghman, R. W., Cowan, C. R., Mih, J. D., Koryakina, Y., Gioeli, D., & Slack-Davis, J. K., et al. (2010). Matrix rigidity regulates cancer cell growth and cellular phenotype. PloS ONE, 5, e12905.
Engler, A. J., Griffin, M. A., Sen, S., Bönnemann, C. G., Sweeney, H. L., & Discher, D. E. (2004). Myotubes differentiate optimally on substrates with tissue-like stiffness pathological implications for soft or stiff microenvironments. The Journal of Cell Biology, 166, 877–887.
Mih, J. D., Sharif, A. S., Liu, F., Marinkovic, A., Symer, M. M., & Tschumperlin, D. J. (2011). A multiwell platform for studying stiffness-dependent cell biology. PloS ONE, 6, e19929.
Engler, A. J., Carag-Krieger, C., Johnson, C. P., Raab, M., Tang, H.-Y., & Speicher, D. W., et al. (2008). Embryonic cardiomyocytes beat best on a matrix with heart-like elasticity: scar-like rigidity inhibits beating. Journal of Cell Science, 121, 3794–3802.
Leach, J. B., Brown, X. Q., Jacot, J. G., DiMilla, P. A., & Wong, J. Y. (2007). Neurite outgrowth and branching of PC12 cells on very soft substrates sharply decreases below a threshold of substrate rigidity. Journal of Neural Engineering, 4, 26.
Lo, C.-M., Wang, H.-B., Dembo, M., & Wang, Y.-l (2000). Cell movement is guided by the rigidity of the substrate. Biophysical Journal, 79, 144–152.
Reilly, G. C., & Engler, A. J. (2010). Intrinsic extracellular matrix properties regulate stem cell differentiation. Journal of Biomechanics, 43, 55–62.
Kloxin, A. M., Benton, J. A., & Anseth, K. S. (2010). In situ elasticity modulation with dynamic substrates to direct cell phenotype. Biomaterials, 31, 1–8.
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.
Carson, D., Hnilova, M., Yang, X., Nemeth, C. L., Tsui, J. H., & Smith, A. S., et al. (2016). Nanotopography-Induced Structural Anisotropy and Sarcomere Development in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells. ACS Applied Materials & Interfaces, 8, 21923–21932.
Tokuda, E. Y., Leight, J. L., & Anseth, K. S. (2014). Modulation of matrix elasticity with PEG hydrogels to study melanoma drug responsiveness. Biomaterials, 35, 4310–4318.
Prauzner-Bechcicki, S., Raczkowska, J., Madej, E., Pabijan, J., Lukes, J., & Sepitka, J., et al. (2015). PDMS substrate stiffness affects the morphology and growth profiles of cancerous prostate and melanoma cells. Journal of the Mechanical Behavior of Biomedical Materials, 41, 13–22.
Agache, P., Monneur, C., Leveque, J., & De Rigal, J. (1980). Mechanical properties and Young’s modulus of human skin in vivo. Archives of Dermatological Research, 269, 221–232.
Pailler-Mattei, C., Bec, S., & Zahouani, H. (2008). In vivo measurements of the elastic mechanical properties of human skin by indentation tests. Medical Engineering & Physics, 30, 599–606.
Budday, S., Nay, R., de Rooij, R., Steinmann, P., Wyrobek, T., & Ovaert, T. C., et al. (2015). Mechanical properties of gray and white matter brain tissue by indentation. Journal of the Mechanical Behavior of Biomedical Materials, 46, 318–330.
Engler, A., Bacakova, L., Newman, C., Hategan, A., Griffin, M., & Discher, D. (2004). Substrate compliance versus ligand density in cell on gel responses. Biophysical Journal, 86, 617–628.
Choi, J. S., & Harley, B. A. (2012). The combined influence of substrate elasticity and ligand density on the viability and biophysical properties of hematopoietic stem and progenitor cells. Biomaterials, 33, 4460–4468.
Jucker, M., Tian, M., & Ingram, D. K. (1996). Laminins in the adult and aged brain. Molecular and Chemical Neuropathology, 28, 209–218.
Ishikawa, T., Wondimu, Z., Oikawa, Y., Gentilcore, G., Kiessling, R., & Brage, S. E., et al. (2014). Laminins 411 and 421 differentially promote tumor cell migration via α6β1 integrin and MCAM (CD146). Matrix Biology, 38, 69–83.
Makishima, A., & Mackenzie, J. (1973). Direct calculation of Young’s moidulus of glass. Journal of Non-Crystalline Solids, 12, 35–45.
Bocchini, V., Mazzolla, R., Barluzzi, R., Blasi, E., Sick, P., & Kettenmann, H. (1992). An immortalized cell line expresses properties of activated microglial cells. Journal of Neuroscience Research, 31, 616–621.
Le Clainche, C., & Carlier, M. F. (2008). Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiological Reviews, 88, 489–513.
Flanagan, L. A., Ju, Y.-E., Marg, B., Osterfield, M., & Janmey, P. A. (2002). Neurite branching on deformable substrates. Neuroreport, 13, 2411.
Buxboim, A., Rajagopal, K., Andre’EX, B., & Discher, D. E. (2010). How deeply cells feel: methods for thin gels. Journal of Physics: Condensed Matter, 22, 194116.
Yeung, T., Georges, P. C., Flanagan, L. A., Marg, B., Ortiz, M., & Funaki, M., et al. (2005). Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motility and the Cytoskeleton, 60, 24–34.
Nakamura, K., Yoshikawa, N., Yamaguchi, Y., Kagota, S., Shinozuka, K., & Kunitomo, M. (2002). Characterization of mouse melanoma cell lines by their mortal malignancy using an experimental metastatic model. Life Sciences, 70, 791–798.
Chen, K., Fu, X., Dorantes-Gonzalez, D. J., Lu, Z., Li, T., & Li, Y., et al. (2014). Simulation study of melanoma detection in human skin tissues by laser-generated surface acoustic waves. Journal of Biomedical Optics, 19, 077007.
Blasi, E., Barluzzi, R., Bocchini, V., Mazzolla, R., & Bistoni, F. (1990). Immortalization of murine microglial cells by a v-raf/v-myc carrying retrovirus. Journal of Neuroimmunology, 27, 229–237.
Chaudhuri, O., Gu, L., Klumpers, D., Darnell, M., Bencherif, S. A., & Weaver, J. C., et al. (2016). Hydrogels with tunable stress relaxation regulate stem cell fate and activity. Nature Materials, 15, 326–334.
Kumar, S., Maxwell, I. Z., Heisterkamp, A., Polte, T. R., Lele, T. P., & Salanga, M., et al. (2006). Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. Biophysical Journal, 90, 3762–3773.
Guilluy, C., Garcia-Mata, R., & Burridge, K. (2011). Rho protein crosstalk: another social network? Trends in Cell Biology, 21, 718–726.
Bangasser, B. L., Shamsan, G. A., Chan, C. E., Opoku, K. N., Tuzel, E., & Schlichtmann, B. W., et al. (2017). Shifting the optimal stiffness for cell migration. Nature Communications, 8, 15313.
Maity, G., Sen, T., & Chatterjee, A. (2011). Laminin induces matrix metalloproteinase-9 expression and activation in human cervical cancer cell line (SiHa). Journal of Cancer Research and Clinical Oncology, 137, 347–357.
Haage, A., & Schneider, I. C. (2014). Cellular contractility and extracellular matrix stiffness regulate matrix metalloproteinase activity in pancreatic cancer cells. FASEB Journal, 28, 3589–3599.
Fligiel, S. E., Laybourn, K. A., Peters, B. P., Ruddon, R. W., Hiserodt, J. C., & Varani, J. (1986). Laminin production by murine melanoma cells: possible involvement in cell motility. Clinical & Experimental Metastasis, 4, 259–272.
Acknowledgements
This work was funded in part by a Research Grant from the Southern Illinois University Edwardsville School of Pharmacy and by start-up funds awarded to Dr. Zustiak by Saint Louis University. Michael Reimer was funded by the Research Grants for Graduate Students program at Southern Illinois University Edwardsville.
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Reimer, M., Petrova Zustiak, S., Sheth, S. et al. Intrinsic Response Towards Physiologic Stiffness is Cell-Type Dependent. Cell Biochem Biophys 76, 197–208 (2018). https://doi.org/10.1007/s12013-017-0834-1
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DOI: https://doi.org/10.1007/s12013-017-0834-1