Annals of Biomedical Engineering

, Volume 45, Issue 6, pp 1399–1406 | Cite as

Proximity of Metastatic Cells Enhances Their Mechanobiological Invasiveness

Article

Abstract

A critical step in metastases formation is cancer-cell invasion through tissue. During invasion, cells change morphology and apply forces to their surroundings. We have previously shown that single, metastatic breast-cancer cells will mechanically indent a synthetic, impenetrable polyacrylamide gel with physiological-stiffness in attempted invasion; benign breast cells do not indent the gels. In solid tumors, e.g., breast cancers, metastases occur predominantly by collective cell-invasion. Thus, here we evaluate the effects of cell proximity on mechanical invasiveness, specifically through changes in gel indention. Gel indentation is induced by 56, 33 and 2% (in >1000 cells), respectively, of adjacent high metastatic potential (MP), low MP and benign breast cells, being double the amounts observed in single, well-separated cells. Single cells exhibited a distribution of indentation depths below 10 µm, while adjacent cells also showed a second peak of deeper indentations. The second peak included 65% of indenting high MP cells as compared to 15% in the low MP cells, illustrating the difference in their invasiveness. Thus, proximity of the metastatic cells enhances their mechanical ability to invade, demonstrating why collective cancer-cell migration is likely more efficient. This could potentially provide a rapid, quantitative approach to identify metastatic cells, and to determine their metastatic potential.

Keywords

Mechanobiology In vitro invasiveness Metastatic potential Cell-substrate mechanical interactions Breast cancer 

Notes

Acknowledgments

The authors thank Mrs. Rakefet Rozen for her assistance in analyzing the results. The work was partially supported by The Technion EVPR Funds—The Elias Fund for Medical Research and The Karbeling Fund for Bio-Medical Engineering Research, and also by a grant from the Ministry of Science, Technology and Space, Israel, and the National Science Council (NSC) of Taiwan.

Supplementary material

10439_2017_1814_MOESM1_ESM.docx (1.7 mb)
Supplementary material 1 (DOCX 1791 kb)

References

  1. 1.
    Abidine, Y., V. Laurent, R. Michel, A. Duperray, L. I. Palade, and C. Verdier. Physical properties of polyacrylamide gels probed by AFM and rheology. Europhys. Lett. 109:38003, 2015.CrossRefGoogle Scholar
  2. 2.
    Abuhattum, S., A. Gefen, and D. Weihs. Ratio of total traction force to projected cell area is preserved in differentiating adipocytes. Integr. Biol. 7:1212–1217, 2015.CrossRefGoogle Scholar
  3. 3.
    Acerbi, I., L. Cassereau, I. Dean, Q. Shi, A. Au, C. Park, Y. Y. Chen, J. Liphardt, E. S. Hwang, and V. M. Weaver. Human breast cancer invasion and aggression correlates with ECM stiffening and immune cell infiltration. Integr. Biol. 7:1120–1134, 2015.CrossRefGoogle Scholar
  4. 4.
    Ahearne, M. Introduction to cell-hydrogel mechanosensing. Interface Focus 4:20130038, 2014.Google Scholar
  5. 5.
    Albini, A., and R. Benelli. The chemoinvasion assay: a method to assess tumor and endothelial cell invasion and its modulation. Nat. Protoc. 2:504–511, 2007.CrossRefPubMedGoogle Scholar
  6. 6.
    Albini, A., Y. Iwamoto, H. K. Kleinman, G. R. Martin, S. A. Aaronson, J. M. Kozlowski, and R. N. McEwan. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 47:3239–3245, 1987.PubMedGoogle Scholar
  7. 7.
    Alvarez-Elizondo, M. B., and D. Weihs. Cell-gel mechanical interactions as an approach to rapidly and quantitatively reveal invasive subpopulations of metastatic cancer cells. Tissue Eng. Part C: Methods 2017. doi: 10.1089/ten.TEC.2016.0424.
  8. 8.
    Boudou, T., J. Ohayon, C. Picart, R. I. Pettigrew, and P. Tracqui. Nonlinear elastic properties of polyacrylamide gels: implications for quantification of cellular forces. Biorheology 46:191–205, 2009.PubMedPubMedCentralGoogle Scholar
  9. 9.
    Butler, J. P., I. M. Tolic-Norrelykke, B. Fabry, and J. J. Fredberg. Traction fields, moments, and strain energy that cells exert on their surroundings. Am. J. Physiol. Physiol. 282:C595–C605, 2002.CrossRefGoogle Scholar
  10. 10.
    Buxboim, A., K. Rajagopal, A. E. X. Brown, and D. E. Discher. How deeply cells feel: methods for thin gels. J. Phys.: Condens. Matter 22(19):194116, 2010.Google Scholar
  11. 11.
    Califano, J. P., and C. A. Reinhart-King. Substrate stiffness and cell area predict cellular traction stresses in single cells and cells in contact. Cell. Mol. Bioeng. 3:68–75, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cheung, K. J., E. Gabrielson, Z. Werb, and A. J. Ewald. Collective invasion in breast cancer requires a conserved basal epithelial program. Cell 155:1639–1651, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Clark, A. G., and D. M. Vignjevic. Modes of cancer cell invasion and the role of the microenvironment. Curr. Opin. Cell Biol. 36:13–22, 2015.CrossRefPubMedGoogle Scholar
  14. 14.
    Cross, S. E., Y. S. Jin, J. Rao, and J. K. Gimzewski. Nanomechanical analysis of cells from cancer patients. Nat. Nanotechnol. 2:780–783, 2007.CrossRefPubMedGoogle Scholar
  15. 15.
    Delanoe-Ayari, H., J. P. Rieu, and M. Sano. 4D traction force microscopy reveals asymmetric cortical forces in migrating dictyostelium cells. Phys. Rev. Lett. 105:248103, 2010.CrossRefPubMedGoogle Scholar
  16. 16.
    Discher, D., C. Dong, J. J. Fredberg, F. Guilak, D. Ingber, P. Janmey, R. D. Kamm, G. W. Schmid-Schonbein, and S. Weinbaum. Biomechanics: cell research and applications for the next decade. Ann. Biomed. Eng. 37:847–859, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Dvir, L., R. Nissim, M. B. Alvarez-Elizondo, and D. Weihs. Quantitative measures to reveal coordinated cytoskeleton-nucleus reorganization during in vitro invasion of cancer cells. New J. Phys. 17:43010, 2015.CrossRefGoogle Scholar
  18. 18.
    Edwards, L. J. Modern statistical techniques for the analysis of longitudinal data in biomedical research. Pediatr. Pulmonol. 30:330–344, 2000.CrossRefPubMedGoogle Scholar
  19. 19.
    Fidler, I. J. The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis. Eur. J. Cancer 9:223–227, 1973.CrossRefPubMedGoogle Scholar
  20. 20.
    Friedl, P., Y. Hegerfeldt, and M. Tusch. Collective cell migration in morphogenesis and cancer. Int. J. Dev. Biol. 48:441–449, 2004.CrossRefPubMedGoogle Scholar
  21. 21.
    Friedl, P., J. Locker, E. Sahai, and J. E. Segall. Classifying collective cancer cell invasion. Nat. Cell Biol. 14:777–783, 2012.CrossRefPubMedGoogle Scholar
  22. 22.
    Friedl, P., and K. Wolf. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat. Rev. Cancer 3:362–374, 2003.CrossRefPubMedGoogle Scholar
  23. 23.
    Friedl, P., and K. Wolf. Tube travel: the role of proteases in individual and collective cancer cell invasion. Cancer Res. 68:7247–7249, 2008.CrossRefPubMedGoogle Scholar
  24. 24.
    Fu, J., Y. K. Wang, M. T. Yang, R. A. Desai, X. Yu, Z. Liu, and C. S. Chen. Mechanical regulation of cell function with geometrically modulated elastomeric substrates. Nat. Methods 7:733–736, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Gal, N., S. Massalha, O. Samuelly-Nafta, and D. Weihs. Effects of particle uptake, encapsulation, and localization in cancer cells on intracellular applications. Med. Eng. Phys. 37:478–483, 2015.CrossRefPubMedGoogle Scholar
  26. 26.
    Gal, N., and D. Weihs. Intracellular mechanics and activity of breast cancer cells correlate with metastatic potential. Cell Biochem. Biophys. 63:199–209, 2012.CrossRefPubMedGoogle Scholar
  27. 27.
    Giannelli, G., J. Falk-Marzillier, O. Schiraldi, W. G. Stetler-Stevenson, and V. Quaranta. Induction of cell migration by matrix metalloprotease-2 cleavage of laminin-5. Science 277:225–228, 1997.CrossRefPubMedGoogle Scholar
  28. 28.
    Goldstein, D., T. Elhanan, M. Aronovitch, and D. Weihs. Origin of active transport in breast-cancer cells. Soft Matter 9:7167–7173, 2013.CrossRefGoogle Scholar
  29. 29.
    Gritsenko, P. G., O. Ilina, and P. Friedl. Interstitial guidance of cancer invasion. J. Pathol. 226:185–199, 2012.CrossRefPubMedGoogle Scholar
  30. 30.
    Guck, J., S. Schinkinger, B. Lincoln, F. Wottawah, S. Ebert, M. Romeyke, D. Lenz, H. M. Erickson, R. Ananthakrishnan, D. Mitchell, J. Kas, S. Ulvick, and C. Bilby. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J . 88:3689–3698, 2005.CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Hur, S. S., Y. H. Zhao, Y. S. Li, E. Botvinick, and S. Chien. Live cells Exert 3-dimensional traction forces on their substrata. Cell. Mol. Bioeng. 2:425–436, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Ilina, O., and P. Friedl. Mechanisms of collective cell migration at a glance. J. Cell Sci. 122:3203–3208, 2009.CrossRefPubMedGoogle Scholar
  33. 33.
    Indra, I., and K. A. Beningo. An in vitro correlation of metastatic capacity, substrate rigidity, and ECM composition. J. Cell. Biochem. 112:3151–3158, 2011.CrossRefPubMedGoogle Scholar
  34. 34.
    Katira, P., R. T. Bonnecaze, and M. H. Zaman. Modeling the mechanics of cancer: effect of changes in cellular and extra-cellular mechanical properties. Front Oncol. 3:145, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Koch, T. M., S. Munster, N. Bonakdar, J. P. Butler, and B. Fabry. 3D Traction forces in cancer cell invasion. PLoS ONE 7:e33476, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Kraning-Rush, C. M., J. P. Califano, and C. A. Reinhart-King. Cellular traction stresses increase with increasing metastatic potential. PLoS ONE 7:e32572, 2012.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Krishnan, R., D. D. Klumpers, C. Y. Park, K. Rajendran, X. Trepat, J. van Bezu, V. W. M. van Hinsbergh, C. V. Carman, J. D. Brain, J. J. Fredberg, J. P. Butler, and G. P. V. Amerongen. Substrate stiffening promotes endothelial monolayer disruption through enhanced physical forces. Am. J. Physiol. Physiol. 300:C146–C154, 2011.CrossRefGoogle Scholar
  38. 38.
    Kristal-Muscal, R., L. Dvir, M. Schvartzer, and D. Weihs. Mechanical interaction of metastatic cancer cells with a soft gel. Procedia IUTAM 12:211–219, 2015.CrossRefGoogle Scholar
  39. 39.
    Kristal-Muscal, R., L. Dvir, and D. Weihs. Metastatic cancer cells tenaciously indent impenetrable, soft substrates. New J. Phys. 15:35022, 2013.CrossRefGoogle Scholar
  40. 40.
    Kumar, S., and V. M. Weaver. Mechanics, malignancy, and metastasis: the force journey of a tumor cell. Cancer Metastasis Rev. 28:113–127, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Lammermann, T., and M. Sixt. Mechanical modes of “amoeboid” cell migration. Curr. Opin. Cell Biol. 21:636–644, 2009.CrossRefPubMedGoogle Scholar
  42. 42.
    Lautscham, L. A. A., C. Kammerer, J. R. R. Lange, T. Kolb, C. Mark, A. Schilling, P. L. L. Strissel, R. Strick, C. Gluth, A. C. C. Rowat, C. Metzner, B. Fabry, C. Kämmerer, J. R. R. Lange, T. Kolb, C. Mark, A. Schilling, P. L. L. Strissel, R. Strick, C. Gluth, A. C. C. Rowat, C. Metzner, and B. Fabry. Migration in confined 3D environments is determined by a combination of adhesiveness, nuclear volume, contractility, and cell stiffness. Biophys. J . 109:900–913, 2015.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Levental, I., P. C. Georges, and P. A. Janmey. Soft biological materials and their impact on cell function. Soft Matter 3:299–306, 2007.CrossRefGoogle Scholar
  44. 44.
    Levental, K. R., H. Yu, L. Kass, J. N. Lakins, M. Egeblad, J. T. Erler, S. F. Fong, K. Csiszar, A. Giaccia, W. Weninger, M. Yamauchi, D. L. Gasser, and V. M. Weaver. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell 139:891–906, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Lo, C. M., H. B. Wang, M. Dembo, and Y. L. Wang. Cell movement is guided by the rigidity of the substrate. Biophys. J . 79:144–152, 2000.CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Maskarinec, S. A., C. Franck, D. A. Tirrell, and G. Ravichandran. Quantifying cellular traction forces in three dimensions. Proc. Natl Acad. Sci. U. S. A. 106:22108–22113, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Massalha, S., and D. Weihs. Metastatic breast cancer cells adhere strongly on varying stiffness substrates, initially without adjusting their morphology. Biomech. Model. Mechanobiol. 2016. doi: 10.1007/s10237-016-0864-4.PubMedGoogle Scholar
  48. 48.
    Menon, S., and K. A. Beningo. Cancer cell invasion is enhanced by applied mechanical stimulation. PLoS ONE 6:e17277, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Oyen, M. L. Mechanical characterisation of hydrogel materials. Int. Mater. Rev. 59:44–59, 2014.CrossRefGoogle Scholar
  50. 50.
    Pankova, K., D. Rosel, M. Novotny, and J. Brabek. The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells. Cell. Mol. Life Sci. 67:63–71, 2010.CrossRefPubMedGoogle Scholar
  51. 51.
    Patsialou, A., J. J. Bravo-Cordero, Y. Wang, D. Entenberg, H. Liu, M. Clarke, and J. S. Condeelis. Intravital multiphoton imaging reveals multicellular streaming as a crucial component of in vivo cell migration in human breast tumors. Intravital 2:e25294, 2013.CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Pelham, R. J., and Y. L. Wang. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl Acad. Sci. U. S. A. 94:13661–13665, 1997.CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Raupach, C., D. P. Zitterbart, C. T. Mierke, C. Metzner, F. A. Muller, and B. Fabry. Stress fluctuations and motion of cytoskeletal-bound markers. Phys. Rev. E 76:11918, 2007.CrossRefGoogle Scholar
  54. 54.
    Sahai, E., and C. J. Marshall. Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nat. Cell Biol. 5:711–719, 2003.CrossRefPubMedGoogle Scholar
  55. 55.
    Sawicki, W., and S. Moskalewski. Hoechst 33342 staining coupled with conventional histological technique. Stain Technol. 64:191–196, 1989.CrossRefPubMedGoogle Scholar
  56. 56.
    Sen, S., A. J. Engler, and D. E. Discher. Matrix strains induced by cells: computing how far cells can feel. Cell. Mol. Bioeng. 2:39–48, 2009.CrossRefPubMedPubMedCentralGoogle Scholar
  57. 57.
    Solon, J., I. Levental, K. Sengupta, P. C. Georges, and P. A. Janmey. Fibroblast adaptation and stiffness matching to soft elastic substrates. Biophys. J . 93:4453–4461, 2007.CrossRefPubMedPubMedCentralGoogle Scholar
  58. 58.
    Stowers, R. S., S. C. Allen, K. Sanchez, C. L. Davis, N. D. Ebelt, C. Van Den Berg, and L. J. Suggs. Extracellular matrix stiffening induces a malignant phenotypic transition in breast epithelial cells. Cell. Mol. Bioeng. 2016. doi: 10.1007/s12195-016-0468-1.Google Scholar
  59. 59.
    Swaminathan, V., K. Mythreye, E. T. O’Brien, A. Berchuck, G. C. Blobe, and R. Superfine. Mechanical stiffness grades metastatic potential in patient tumor cells and in cancer cell lines. Cancer Res. 71:5075–5080, 2011.CrossRefPubMedPubMedCentralGoogle Scholar
  60. 60.
    Trepat, X., B. Fabry, and J. J. Fredberg. Pulling it together in three dimensions. Nat. Methods 7:963–965, 2010.CrossRefPubMedPubMedCentralGoogle Scholar
  61. 61.
    Wagoner Johnson, A., and B. A. Harley. Mechanobiology of Cell–Cell and Cell–Matrix Interactions. New York: Springer, p. 319, 2011.CrossRefGoogle Scholar
  62. 62.
    Weston, S. A., and C. R. Parish. New fluorescent dyes for lymphocyte migration studies. Analysis by flow cytometry and fluorescence microscopy. J. Immunol. Methods 133:87–97, 1990.CrossRefPubMedGoogle Scholar
  63. 63.
    Wolf, K., Y. I. Wu, Y. Liu, J. Geiger, E. Tam, C. Overall, M. S. Stack, and P. Friedl. Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat. Cell Biol. 9:893–904, 2007.CrossRefPubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2017

Authors and Affiliations

  1. 1.Faculty of Biomedical EngineeringTechnion-Israel Institute of TechnologyHaifaIsrael

Personalised recommendations