BioNanoScience

, Volume 6, Issue 1, pp 54–64 | Cite as

Silencing of CD44 in Glioma Leads to Changes in Cytoskeletal Protein Expression and Cellular Biomechanical Deformation Properties as Measured by AFM Nanoindentation

  • Zaynah Maherally
  • James R. Smith
  • Manar K. Ghoneim
  • Luke Dickson
  • Qian An
  • Helen L. Fillmore
  • Geoffrey J. Pilkington
Article

Abstract

CD44, a transmembrane glycoprotein receptor for extracellular matrix molecules such as hyaluronic acid and osteopontin, is involved in glioma cellular signalling, adhesion and invasion. Although a great deal is known concerning the molecular players in adhesion, migration and invasion, little is known relating to how these invasive and migratory-promoting proteins influence biomechanical properties of glioma cells. Herein, we extend previous CD44 blocking experiments to examine effects of CD44 knock-down on expression of cytoskeletal proteins and cellular stiffness. An atomic force microscope (AFM) nanoindentation method was used to measure deformation or cellular stiffness (Young’s modulus, E) in real time, at the single-cell level over nuclear and cytoplasmic regions. A glioblastoma cell line (SNB-19) was transfected with either CD44 small interfering RNA (siRNA), scrambled siRNA or a non-related gene siRNA. In SNB-19 CD44 knock-down cells, levels of microtubule, vimentin and glial fibrillary acidic protein (GFAP) proteins were lower compared to cells transfected with scrambled siRNA. Functionally, CD44 knock-down cells were less migratory compared to controls. AFM nanoindentation results show that the areas over the nuclei of both knock-down and parental control cells examined were significantly more compliant than their cytoplasmic regions (p < 0.001). The most striking difference was seen when comparing nuclear regions of parental control cells versus CD44 knock-down cells. CD44 knock-down SNB-19 cells (E = 0.56 ± 0.50 kPa) were less stiff than parental cells (E = 1.93 ± 2.86 kPa; p < 0.001). Based on these results, we hypothesise that CD44 signalling via cytoskeletal proteins such as vimentin may influence the ability of glioma cells to respond to host tumour-derived mechanical pressures.

Keywords

Glioma Migration Cytoskeleton Cell stiffness Atomic force microscopy (AFM) 

Notes

Acknowledgments

We thank Drs Robert Field and Alex Winkle from JPK Instruments, Cambridge, UK, for loan of the CellHesion 200 and NanoWizard 3 AFM instruments and Dr Torsten Muller from JPK Instruments, Berlin, Germany, for initial AFM training. We also thank Brain Tumour Research and Institute for Biomedical and Biomolecular Sciences for support.

References

  1. 1.
    Rape, A., Ananthanarayanan, B., Kumar, S. (2014). Engineering strategies to mimic the glioblastoma microenvironment. Advanced Drug Delivery Reviews, 79–80, 172–183.CrossRefGoogle Scholar
  2. 2.
    Xie, Q., Mittal, S., Berens, M. E. (2014). Targeting adaptive glioblastoma: an overview of proliferation and invasion. Neuro-Oncology, 16, 1575–1584.CrossRefGoogle Scholar
  3. 3.
    Kim, Y., & Kumar, S. (2014). CD44-mediated adhesion to hyaluronic acid contributes to mechanosensing and invasive motility. Molecular Cancer Research, 12, 1416–1429.CrossRefGoogle Scholar
  4. 4.
    Ketene, A. N., Schmelz, E. M., Roberts, P. C., Agah, M. (2012). The effects of cancer progression on the viscoelasticity of ovarian cell cytoskeleton structures. Nanomedicine, 8, 93–102.CrossRefGoogle Scholar
  5. 5.
    Babahosseini, H., Ketene, A. N., Schmelz, E. M., Roberts, P. C., Agah, M. (2014). Biomechanical profile of cancer stem-like/tumor-initiating cells derived from a progressive ovarian cancer model. Nanomedicine, 10, 1013–1019.CrossRefGoogle Scholar
  6. 6.
    Lekka, M., Gil, D., Pogoda, K., Dulińska-Litewka, J., Jach, R., Gostek, J., et al. (2012). Cancer cell detection in tissue sections using AFM. Archives of Biochemistry and Biophysics, 518, 151–156.CrossRefGoogle Scholar
  7. 7.
    Swaminathan, V., Mythreye, K., O’Brien, E. T., Berchuck, A., Blobe, G. C., Superfine, R. (2011). Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Research, 71, 5075–5080.CrossRefGoogle Scholar
  8. 8.
    Yamazaki, D., Kurisu, S., Takenawa, T. (2005). Regulation of cancer cell motility through actin reorganization. Cancer Science, 96, 379–386.CrossRefGoogle Scholar
  9. 9.
    Rao, J., & Li, N. (2004). Microfilament actin remodeling as a potential target for cancer drug development. Current Cancer Drug Targets, 4, 345–354.CrossRefGoogle Scholar
  10. 10.
    Weder, G., Hendriks-Balk, M. C., Smajda, R., Rimoldi, D., Liley, M., Heinzelmann, H., et al. (2014). Increased plasticity of the stiffness of melanoma cells correlates with their acquisition of metastatic properties. Nanomedicine, 10, 141–148.CrossRefGoogle Scholar
  11. 11.
    McKnight, A. L., Kugel, J. L., Rossman, P. J., Manduca, A., Hartmann, L. C., Ehman, R. L. (2002). MR elastography of breast cancer: preliminary results. AJR. American Journal of Roentgenology, 178, 1411–1417.CrossRefGoogle Scholar
  12. 12.
    Bercoff, J., Chaffai, S., Tanter, M., Sandrin, L., Catheline, S., Fink, M., et al. (2003). In vivo breast tumor detection using transient elastography. Ultrasound in Medicine and Biology, 29, 1387–1396.CrossRefGoogle Scholar
  13. 13.
    Suresh, S. (2007). Biomechanics and biophysics of cancer cells. Acta Biomaterialia, 3, 413–438.MathSciNetCrossRefGoogle Scholar
  14. 14.
    Birch, M., Mitchell, S., Hart, I. R. (1991). Isolation and characterization of human melanoma call variants expressing high and low levels of CD44. Cancer Research, 51, 6660–6667.Google Scholar
  15. 15.
    Sy, M. S., Guo, Y. J., Stamenkovic, I. (1991). Distinct effects of two CD44 isoforms on tumour growth in vivo. Journal of Experimental Medicine, 174, 859–866.CrossRefGoogle Scholar
  16. 16.
    Okada, H., Yoshida, J., Sokabe, M., Wakabayashi, T., Hagiwara, M. (1991). Supression of CD44 expression decreases migration and invasion of human glioma cells. International Journal of Cancer, 66, 255–260.CrossRefGoogle Scholar
  17. 17.
    Gunia, S., Hussein, S., Radu, D. L., Putz, K. M., Breyer, R., Hecker, H. (1999). CD44s-targeted treatment with monoclonal antibody blocks intercerebral invasion and growth of 9L gliosarcoma. Clinical and Experimental Metastasis, 17, 221–230.CrossRefGoogle Scholar
  18. 18.
    Stern, R., Shuster, S., Wiley, T. S., Formby, B. (2001). Hyaluronidase can modulate expression of CD44. Experimental Cell Research, 266, 167–319.CrossRefGoogle Scholar
  19. 19.
    Wiranowska, M., Ladd, S., Moscinski, L. C., Hill, B., Haller, E., Mikecz, K., et al. (2010). Modulation of hyaluronan production by CD44 positive glioma cells. International Journal of Cancer, 127, 532–542.CrossRefGoogle Scholar
  20. 20.
    Pietras, A., Katz, A. M., Ekström, E. J., Wee, B., Halliday, J. J., Pitter, K. L., et al. (2014). Osteopontin-CD44 signaling in the glioma perivascular niche enhances cancer stem cell phenotypes and promotes aggressive tumor growth. Cell Stem Cell, 14, 357–369.CrossRefGoogle Scholar
  21. 21.
    Misra, S., Hascall, V. C., De Giovanni, C., Markwald, R. R., Ghatak, S. (2009). Delivery of CD44 shRNA/nanoparticles within cancer cells. Journal of Biological Chemistry, 284, 12432–12446.CrossRefGoogle Scholar
  22. 22.
    Stamenkovic, I., Aruffo, A., Amiot, M. (1991). The hematopoietic and epithelial forms of CD44 are distinct polypeptides with different adhesion potentials for hyaluronate-bearing cells. EMBO Journal, 10, 343–348.Google Scholar
  23. 23.
    Delpech, B., Maingonnat, C., Girard, N., Chauzy, C., Maunoury, R., Olivier, A. (1993). Hyaluronan and hyaluronectic in the extracellular matrix of human brain tumour stroma. European Journal of Cancer, 29A, 1012–1017.CrossRefGoogle Scholar
  24. 24.
    Merzak, A., & Pilkington, G. J. (1997). Molecular and cellular pathology of intrinsic brain tumours. Cancer and Metastasis Reviews, 16, 155–177.CrossRefGoogle Scholar
  25. 25.
    Günthert, U., Hofmann, M., Rudy, W., Reber, S., Zöller, M., Haussmann, I., et al. (1991). A new variant of glycoprotein CD44 confers metastatic potential to rat carcinoma cells. Cell, 65, 13–24.CrossRefGoogle Scholar
  26. 26.
    Okamoto, I., Tsuiki, H., Kenyon, L. C., Godwin, A. K., Emlet, D. R. (2002). Proteolytic cleavage of the CD44 adhesion molecule in multiple human tumours. American Journal of Pathology, 160, 441–447.CrossRefGoogle Scholar
  27. 27.
    Maherally, Z., Smith, J. R., An, Q., Pilkington, G. J. (2012). Receptors for hyaluronic acid and poliovirus: a combinatorial role in glioma invasion? PLoS One, 7, e30691.CrossRefGoogle Scholar
  28. 28.
    Mackay, J. L., Keung, A. J., Kumar, S. (2012). A genetic strategy for the dynamic and graded control of cell mechanics, motility, and matrix remodelling. Biophysical Journal, 102, 434–442.CrossRefGoogle Scholar
  29. 29.
    Wang, B., Lancon, P., Bienvenu, C., Vierling, P., Di Giorgio, C., Bossis, G. (2013). A general approach for the microrheology of cancer cells by atomic force microscopy. Micron, 44, 287–297.CrossRefGoogle Scholar
  30. 30.
    Vadillo-Rodriguez, V., & Dutcher, J. R. (2009). Dynamic viscoelastic behavior of individual Gram-negative bacterial cells. Soft Matter, 5, 5012–5019.CrossRefGoogle Scholar
  31. 31.
    Eaton, P., & West, P. (2010). Atomic force microscopy. Oxford: Oxford Univ Press.CrossRefGoogle Scholar
  32. 32.
    Binnig, G., Quate, C. F., Gerber, C. (1986). Atomic force microscope. Physical Review Letters, 56, 930–933.CrossRefGoogle Scholar
  33. 33.
    Sokolov, I., Iyer, S., Woodworth, C. D. (2006). Recovery of elasticity of aged human epithelial cells in vitro. Nanomedicine: Nanotechnology, Biology and Medicine, 2, 31–36.CrossRefGoogle Scholar
  34. 34.
    Finke, M., Hughes, J. A., Parker, D. M., Jandt, K. D. (2001). Mechanical properties of in situ demineralised human enamel measured by AFM nanoindentation. Surface Science, 491, 456–467.CrossRefGoogle Scholar
  35. 35.
    Lekka, M., Laidler, P., Gil, D., Lekki, J., Stachura, Z., Hrynkiewicz, A. Z. (1999). Elasticity of normal and cancerous human bladder cells studied by scanning force microscopy. European Biophysics Journal, 28, 312–316.CrossRefGoogle Scholar
  36. 36.
    Chen, B., Wang, Q., Han, L. (2004). Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer. Scanning, 26, 162–166.CrossRefGoogle Scholar
  37. 37.
    Cross, S. E., Jin, Y. S., Rao, J., Gimzewski, J. K. (2007). Nanomechanical analysis of cells from cancer patients. Nature Nanotechnology, 2, 780–783.CrossRefGoogle Scholar
  38. 38.
    Cross, S. E., Jin, Y. S., Tondre, J., Wong, R., Rao, J., Gimzewski, J. K. (2008). AFM-based analysis of human metastatic cancer cells. Nanotechnology, 19, 384003.CrossRefGoogle Scholar
  39. 39.
    Li, Q. S., Lee, G. Y., Ong, C. N., Lim, C. T. (2008). AFM indentation study of breast cancer cells. Biochemical and Biophysical Research Communications, 374, 609–613.CrossRefGoogle Scholar
  40. 40.
    Plodinec, M., Loparic, M., Monnier, C. A., Obermann, E. C., Zanetti-Dallenbach, R., Oertle, P., et al. (2012). The nanomechanical signature of breast cancer. Nature Nanotechnology, 7, 757–765.CrossRefGoogle Scholar
  41. 41.
    Yokokawa, M. K., Takeyasu, Yoshimura, S. H. (2008). Mechanical properties of plasma membrane and nuclear envelope measured by scanning probe microscope. Journal of Microscopy, 232, 82–90.MathSciNetCrossRefGoogle Scholar
  42. 42.
    Fuhrmann, A., Staunton, J. R., Nandakumar, V., Banyai, N., Davies, P. C. W., Ros, R. (2011). AFM stiffness nanotomography of normal, metaplastic and dysplastic human esophageal cells. Physical Biology, 8, 015007.CrossRefGoogle Scholar
  43. 43.
    Zhang, G., Long, M., Wu, Z. Z., Yu, W. Q. (2002). Mechanical properties of hepatocellular carcinoma cells. World Journal of Gastroenterology, 8, 243–246.Google Scholar
  44. 44.
    Gang, Z., Qi, Q., Jing, C., Wang, C. Y. (2009). Measuring microenvironment mechanical stress of rat liver during diethylnitrosamine induced hepatocarcinogenesis by atomic force microscope. Microscopy Research and Technique, 72, 672–678.CrossRefGoogle Scholar
  45. 45.
    Xu, W., Mezencev, R., Kim, B., Wang, L., McDonald, J., Sulchek, T. (2012). Cell stiffness is a biomarker of the metastatic potential of ovarian cancer cells. PLoS One, 7(10), e46609.CrossRefGoogle Scholar
  46. 46.
    Li, Y. X., Schnekenburger, J., Duits, M. H. G. (2009). Intracellular particle tracking as a tool for tumor cell characterization. Journal Biomedical Optics, 14, 064005.CrossRefGoogle Scholar
  47. 47.
    Moreno-Flores, S., Benitez, R., Vivanco, M. D., Toca-Herrera, J. L. (2010). Stress relaxation and creep on living cells with the atomic force microscope: a means to calculate elastic moduli and viscosities of cell components. Nanotechnology, 21, 445101.CrossRefGoogle Scholar
  48. 48.
    Faria, E. C., Ma, N., Gazi, E., Gardner, P., Brown, M., Noel, N. W., et al. (2008). Measurement of elastic properties of prostate cancer cells using AFM. Analyst, 133, 1498–1500.CrossRefGoogle Scholar
  49. 49.
    Docheva, D., Padula, D., Schieker, M., Clausen-Schaumann, H. (2010). Effect of collagen I and fibronectin on the adhesion, elasticity and cytoskeletal organization of prostate cancer cells. Biochemical and Biophysical Research Communications, 402, 361–366.CrossRefGoogle Scholar
  50. 50.
    Prabhune, M., Belge, G., Dotzauer, A., Bullerdiek, J., Radmacher, M. (2012). Comparison of mechanical properties of normal and malignant thyroid cells. Micron, 43, 1267–1272.CrossRefGoogle Scholar
  51. 51.
    An, Q., Fillmore, H. L., Vouri, M., Pilkington, G. J. (2014). Brain tumour cell line authentication, an efficient alternative to capillary electrophoresis by using a microfluidics-based system. Neuro-Oncology, 16, 265–273.CrossRefGoogle Scholar
  52. 52.
    Clifford, C. A., & Seah, M. P. (2005). Quantification issues in the identification of nanoscale regions of homopolymers using modulus measurement via AFM nanoindentation. Applied Surface Science, 252, 1915–1933.CrossRefGoogle Scholar
  53. 53.
    Hutter, J. L., & Bechhoefer, J. (1993). Calibration of atomic-force microscope tips. Review of Scientific Instruments, 64, 1868–1873.CrossRefGoogle Scholar
  54. 54.
    McPhee, G., Dalby, M. J., Riehle, M., Yin, H. B. (2010). Can common adhesion molecules and microtopography affect cellular elasticity? A combined atomic force microscopy and optical study. Medical and Biological Engineering and Computing, 48, 1043–1053.CrossRefGoogle Scholar
  55. 55.
    Hertz, H. (1881). Ueber die Berührung fester elastischer Körper. Journal für die Reine und Angewandte Mathematik, 92, 156–171.MathSciNetGoogle Scholar
  56. 56.
    Touham, A., Nysten, B., Dufrene, Y. F. (2003). Nanoscale mapping of the elasticity of microbial cells by atomic force microscopy. Langmuir, 19, 4539–4543.CrossRefGoogle Scholar
  57. 57.
    Dimitriadis, E. K., Horkay, F., Maresca, J., Kachar, B., Chadwick, R. S. (2002). Determination of elastic moduli of thin layers of soft material using the atomic force microscope. Biophysical Journal, 82, 2798–2810.CrossRefGoogle Scholar
  58. 58.
    Pett, M. A. (1997). Nonparametric statistics for health care research. Thousand Oaks: SAGE Publications Inc.Google Scholar
  59. 59.
    Herpers, M. J., Budka, H., McCormick, D. (1984). Production of glial fibrillary acidic protein (GFAP) by neoplastic cells: adaptation to the microenvironment. Acta Neuropathologica, 64, 333–338.CrossRefGoogle Scholar
  60. 60.
    Rathje, L. Z., Nordgren, N., Pettersson, T., Rönnlund, D., Widengren, J., Aspenström, P., et al. (2014). Oncogenes induce a vimentin filament collapse mediated by HDAC6 that is linked to cell stiffness. Proceedings of the National Academy of Sciences of the United States of America, 11, 1515–1520.CrossRefGoogle Scholar
  61. 61.
    Chang, L., & Goldman, R. D. (2004). Intermediate filaments mediate cytoskeletal crosstalk. Molecular and Cellular Biology, 5, 601–613.Google Scholar
  62. 62.
    Tai, P. W., Zaidi, S. K., Wu, H., Grandy, R. A., Montecino, M., van Wijnen, A. J., et al. (2014). The dynamic architectural and epigenetic nuclear landscape: developing the genomic almanac of biology and disease. Journal of Cellular Physiology, 229, 711–727.CrossRefGoogle Scholar
  63. 63.
    Gorjanacz, M. (2014). Nuclear assembly as a target for anti-cancer therapies. Nucleus, 5, 47–55.CrossRefGoogle Scholar
  64. 64.
    Schreiner, S. M., Koo, P. K., Zhao, Y., Mochrie, S. G. J., King, M. C. (2015). The tethering of chromatin to the nuclear envelope supports nuclear mechanics. Nature Communications, 6, 7159–7171.CrossRefGoogle Scholar
  65. 65.
    Osmanagic-Myers, S., Dechat, T., Foisner, R. (2015). Lamins at the crossroads of mechanosignaling. Genes and Development, 29, 225–237.CrossRefGoogle Scholar
  66. 66.
    Fedorchak, G. R., Kaminski, A., Lammerding, J. (2014). Cellular mechanosensing: getting to the nucleus of it all. Progress in Biophysics and Molecular Biology, 115, 76–92.CrossRefGoogle Scholar
  67. 67.
    Swift, J., & Discher, D. E. (2014). The nuclear lamina is mechano-responsive to ECM elasticity in mature tissue. Journal of Cell Science, 127, 3005–3015.CrossRefGoogle Scholar
  68. 68.
    Harada, T., Swift, J., Irianto, J., Shin, J.-W., Spinler, K. R., Athirasala, A., et al. (2014). Nuclear lamin stiffness is a barrier to 3D migration, but softness can limit survival. Journal of Cell Biology, 204, 669–682.CrossRefGoogle Scholar
  69. 69.
    Schape, J., Prauße, S., Radmacher, M., Stick, R. (2009). Influence of lamin A on the mechanical properties of amphibian oocyte nuclei measured by atomic force microscopy. Biophysical Journal, 96, 4319–4325.CrossRefGoogle Scholar
  70. 70.
    Ferrera, D., Canale, C., Marotta, R., Mazzaro, N., Gritti, M., Mazzanti, M., et al. (2014). Lamin B1 overexpression increases nuclear rigidity in autosomal dominant leukodystrophy fibroblasts. FASEB Journal, 28, 3906–3918.CrossRefGoogle Scholar
  71. 71.
    Ramos, J. R., Rabijan, J., Garcia, R., Lekka, M. (2014). The softening of human bladder cancer cells happens at an early stage of the malignancy process. Beilstein Journal of Nanotechnology, 5, 447–457.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Zaynah Maherally
    • 1
  • James R. Smith
    • 1
  • Manar K. Ghoneim
    • 1
  • Luke Dickson
    • 1
  • Qian An
    • 1
  • Helen L. Fillmore
    • 1
  • Geoffrey J. Pilkington
    • 1
  1. 1.Cellular and Molecular Neuro-oncology Research Group, Institute of Biomedical and Biomolecular Sciences, School of Pharmacy and Biomedical SciencesUniversity of PortsmouthPortsmouthUK

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