Label-Free Automated Cell Tracking: Analysis of the Role of E-cadherin Expression in Collective Electrotaxis


Collective cell migration plays an important role in wound healing, organogenesis, and the progression of metastatic disease. Analysis of collective migration typically involves laborious and time-consuming manual tracking of individual cells within cell clusters over several dozen or hundreds of frames. Herein, we develop a label-free, automated algorithm to identify and track individual epithelial cells within a free-moving cluster. We use this algorithm to analyze the effects of partial E-cadherin knockdown on collective migration of MCF-10A breast epithelial cells directed by an electric field. Our data show that E-cadherin knockdown in free-moving cell clusters diminishes electrotactic potential, with empty vector MCF-10A cells showing 16% higher directedness than cells with E-cadherin knockdown. Decreased electrotaxis is also observed in isolated cells at intermediate electric fields, suggesting an adhesion-independent role of E-cadherin in regulating electrotaxis. In additional support of an adhesion-independent role of E-cadherin, isolated cells with reduced E-cadherin expression reoriented within an applied electric field 60% more quickly than control. These results have implications for the role of E-cadherin expression in electrotaxis and demonstrate proof-of-concept of an automated algorithm that is broadly applicable to the analysis of collective migration in a wide range of physiological and pathophysiological contexts.

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  1. 1.

    Aftab, O., M. Fryknäs, U. Hammerling, R. Larsson, and M. G. Gustafsson. Detection of cell aggregation and altered cell viability by automated label-free video microscopy: A promising alternative to endpoint viability assays in high- throughput screening. J. Biomol. Screen. 20:372–381, 2015.

    Article  Google Scholar 

  2. 2.

    Ahrens, E. T., and J. Zhong. In vivo MRI cell tracking using perfluorocarbon probes and fluorine-19 detection. NMR Biomed. 26:860–871, 2013.

    Article  Google Scholar 

  3. 3.

    Anon, E., X. Serra-Picamal, P. Hersen, N. C. Gauthier, M. P. Sheetz, X. Trepat, and B. Ladoux. Cell crawling mediates collective cell migration to close undamaged epithelial gaps. Proc. Natl. Acad. Sci. 109:10891–10896, 2012.

    Article  Google Scholar 

  4. 4.

    Arora, A., and T. Qazi. Computer vision based tracking of biological cells: A review. Int. Conf. Adv. Res. Innov. 118–126, 2014.

  5. 5.

    Bengtsson, E., C. Wahlby, and J. Lindblad. Robust Cell Image segmentation methods. Pattern Recognit. Image Anal. 14:157–167, 2004.

    Google Scholar 

  6. 6.

    Bradley, M. O., and N. A. Sharkey. Metagenicity and toxicity of visivle fluorescent light to cultured mammalian cells. Nature 267:673–678, 1977.

    Article  Google Scholar 

  7. 7.

    Cai, D., S. C. Chen, M. Prasad, L. He, X. Wang, V. Choesmel-Cadamuro, J. K. Sawyer, G. Danuser, and D. J. Montell. Mechanical feedback through E-cadherin promotes direction sensing during collective cell migration. Cell 157:1146–1159, 2014.

    Article  Google Scholar 

  8. 8.

    Carey, S. P., A. Starchenko, A. L. McGregor, and C. A. Reinhart-King. Leading malignant cells initiate collective epithelial cell invasion in a three-dimensional heterotypic tumor spheroid model. Clin. Exp. Metastasis 30:615–630, 2013.

    Article  Google Scholar 

  9. 9.

    Chalfound, J., M. Kociolek, A. Dima, M. Halter, A. Cardone, A. Peskin, P. Bajcsy, and M. Brady. Segmenting time-lapse phase contrast images of adjacent NIH 3T3 cells. J. Microsc. 249:41–52, 2013.

    Article  Google Scholar 

  10. 10.

    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.

    Article  Google Scholar 

  11. 11.

    Cuzick, J., R. Holland, V. Barth, R. Davies, M. Faupel, I. Fentiman, H. J. Frischbier, J. L. LaMarque, M. Merson, V. Sacchini, D. Vanel, and U. Veronesi. Electropotential measurements as a new diagnostic modality for breast cancer. Lancet 352:359–363, 1998.

    Article  Google Scholar 

  12. 12.

    Débarre, D., and E. Beaurepaire. Quantitative characterization of biological liquids for third-harmonic generation microscopy. Biophys. J. 92:603–612, 2007.

    Article  Google Scholar 

  13. 13.

    Fogg, V. C., C.-J. Liu, and B. Margolis. Multiple regions of Crumbs3 are required for tight junction formation in MCF10A cells. J. Cell Sci. 118:2859–2869, 2005.

    Article  Google Scholar 

  14. 14.

    Friedl, P., and D. Gilmour. Collective cell migration in morphogenesis, regeneration and cancer. Nat. Rev. Mol. Cell Biol. 10:445–457, 2009.

    Article  Google Scholar 

  15. 15.

    Friedl, P., J. Locker, E. Sahai, and J. E. Segall. Classifying collective cancer cell invasion. Nat. Cell Biol. 14:777–783, 2012.

    Article  Google Scholar 

  16. 16.

    Graham, N. A., and A. R. Asthagiri. Epidermal growth factor-mediated T-cell factor/lymphoid enhancer factor transcriptional activity is essential but not sufficient for cell cycle progression in nontransformed mammary epithelial cells. J. Biol. Chem. 279:23517–23524, 2004.

    Article  Google Scholar 

  17. 17.

    Hazan, R. B., and L. Norton. The epidermal growth factor receptor modulates the interaction of E-cadherin with the actin cytoskeleton. J Biol Chem 273:9078–9084, 1998.

    Article  Google Scholar 

  18. 18.

    Jaccard, N., N. Szita, and L. D. Griffin. Computer methods in biomechanics and biomedical engineering: Imaging and visualization segmentation of phase contrast microscopy images based on multi-scale local Basic Image Features histograms. Comput. Methods Biomech. Biomed. Eng. Imaging Vis. 1–9, 2015.

  19. 19.

    Kushiro, K., and A. R. Asthagiri. Modular design of micropattern geometry achieves combinatorial enhancements in cell motility. Langmuir 28:4357–4362, 2012.

    Article  Google Scholar 

  20. 20.

    Lalli, M. L., and A. R. Asthagiri. Collective migration exhibits greater sensitivity but slower dynamics of alignment to applied electric fields. Cell. Mol. Bioeng. 8:247–257, 2015.

    Article  Google Scholar 

  21. 21.

    Latt, S. A. Optical studies of metaphase chromosome organization. Clin. Genet. 19:154–161, 1976.

    Google Scholar 

  22. 22.

    Li, L., R. Hartley, B. Reiss, Y. Sun, J. Pu, D. Wu, F. Lin, T. Hoang, S. Yamada, J. Jiang, and M. Zhao. E-cadherin plays an essential role in collective directional migration of large epithelial sheets. Cell. Mol. Life Sci. 69:2779–2789, 2012.

    Article  Google Scholar 

  23. 23.

    Li, X., and J. Kolega. Effects of direct current electric fields on cell migration and actin filament distribution in bovine vasclar endothelial cells. J. Vasc. Res. 39:391–404, 2002.

    Article  Google Scholar 

  24. 24.

    Marrison, J., L. Räty, P. Marriott, and P. O’Toole. Ptychography–a label free, high-contrast imaging technique for live cells using quantitative phase information. Sci. Rep. 3:2369, 2013.

    Article  Google Scholar 

  25. 25.

    McCaig, C. D., B. Song, and A. M. Rajnicek. Electrical dimensions in cell science. J. Cell Sci. 122:4267–4276, 2009.

    Article  Google Scholar 

  26. 26.

    Meijering, E. Cell segmentation: 50 years down the road. IEEE Signal Process. Mag. 29:140–145, 2012.

    Article  Google Scholar 

  27. 27.

    Milano, D. F., N. A. Ngai, S. K. Muthuswamy, and A. R. Asthagiri. Regulators of metastasis modulate the migratory response to cell contact under spatial confinement. Biophys. J. 110:1886–1895, 2016.

    Article  Google Scholar 

  28. 28.

    Mousavi, S. J., M. H. Doweidar, and M. Doblaré. 3D computational modelling of cell migration: A mechano-chemo-thermo-electrotaxis approach. J. Theor. Biol. Elsevier 329:64–73, 2013.

    Article  MATH  Google Scholar 

  29. 29.

    Ng, M. R., A. Besser, G. Danuser, and J. S. Brugge. Substrate stiffness regulates cadherin-dependent collective migration through myosin-II contractility. J. Cell Biol. 199:545–563, 2012.

    Article  Google Scholar 

  30. 30.

    Oka, H., H. Shiozaki, K. Kobayashi, M. Inoue, H. Tahara, T. Kobayashi, Y. Takatsuka, N. Matsuyoshi, S. Hirano, M. Takeichi, and T. Mori. Expression of E-cadherin cell adhesion molecules in human breast cancer tissues and its relationship to metastasis. Cancer Res. 53:1696–1701, 1993.

    Google Scholar 

  31. 31.

    Olivier, N., M. A. Luengo-oroz, L. Duloquin, E. Faure, T. Savy, I. Veilleux, X. Solinas, D. Débarre, P. Bourgine, A. Santos, N. Peyriéras, and E. Beaurepaire. Cell Lineage Reconstr. Early. 70, 2007.

  32. 32.

    Onder, T. T., P. B. Gupta, S. A. Mani, J. Yang, E. S. Lander, and R. A. Weinberg. Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways. Cancer Res. 68:3645–3654, 2008.

    Article  Google Scholar 

  33. 33.

    Progatzky, F., M. J. Dallman, and C. Lo Celso. From seeing to believing: labelling strategies for in vivo cell-tracking experiments. Interface Focus 3:1–14, 2013.

    Article  Google Scholar 

  34. 34.

    Pu, J., C. D. McCaig, L. Cao, Z. Zhao, J. E. Segall, and M. Zhao. EGF receptor signalling is essential for electric-field-directed migration of breast cancer cells. J. Cell Sci. 120:3395–3403, 2007.

    Article  Google Scholar 

  35. 35.

    Rodriguez, L. L., and I. C. Schneider. Directed cell migration in multi-cue environments. Integr. Biol. 5:1306–1323, 2013.

    Article  Google Scholar 

  36. 36.

    Rompolas, P., E. R. Deschene, G. Zito, D. G. Gonzalez, I. Saotome, A. M. Haberman, and V. Greco. Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration. Nature 487:496–499, 2012.

    Article  Google Scholar 

  37. 37.

    Sandquist, J. C., K. I. Swenson, K. A. Demali, K. Burridge, and A. R. Means. Rho kinase differentially regulates phosphorylation of nonmuscle myosin II isoforms A and B during cell rounding. J. Biol. Chem. 281:35873–35883, 2006.

    Article  Google Scholar 

  38. 38.

    Sbalzarini, I., and P. Koumoutsakos. Feature point tracking and trajectory analysis for video imaging in cell biology. J. Struct. Biol. 151:182–195, 2005.

    Article  Google Scholar 

  39. 39.

    Schindelin, J., I. Arganda-Carreras, E. Frise, V. Kaynig, M. Longair, T. Pietzsch, S. Preibisch, C. Rueden, S. Saalfeld, B. Schmid, J.-Y. Tinevez, D. J. White, V. Hartenstein, K. Liceiri, P. Tomancak, and A. Cardona. Fiji: an open source platform for biological image analysis. Nat. Methods 9:676–682, 2012.

    Article  Google Scholar 

  40. 40.

    Schmalhofer, O., S. Brabletz, and T. Brabletz. E-cadherin, β-catenin, and ZEB1 in malignant progression of cancer. Cancer Metastasis Rev. 28:151–166, 2009.

    Article  Google Scholar 

  41. 41.

    Sommers, C., E. Gelmann, C. L. Sommers, E. W. Thompson, R. Kemlen, E. P. Gelmann, S. W. Byers, and A. Torn. Cell adhesion molecule uvomorulin expression in human breast cancer cell lines: Relationship to morphology and invasive capacities Uvomorulin Breast Cancer Cell Lines: Relationship and Invasive in Human to Morphology. Cell Growth Differ. 2:365–372, 1991.

    Google Scholar 

  42. 42.

    Song, B., Y. Gu, J. Pu, B. Reid, Z. Zhao, and M. Zhao. Application of direct current electric fields to cells and tissues in vitro and modulation of wound electric field in vivo. Nat. Protoc. 2:1479–1489, 2007.

    Article  Google Scholar 

  43. 43.

    Tsai, H. F., C. W. Huang, H. F. Chang, J. J. W. Chen, C. H. Lee, and J. Y. Cheng. Evaluation of EGFR and RTK signaling in the electrotaxis of lung adenocarcinoma cells under direct-current electric field stimulation. PLoS ONE 8:1–20, 2013.

    Google Scholar 

  44. 44.

    Van Roy, F., and G. Berx. The cell-cell adhesion molecule E-cadherin. Cell. Mol. Life Sci. 65:3756–3788, 2008.

    Article  Google Scholar 

  45. 45.

    Veit, W., C. Held, R. Palmisano, and T. Wittenberg. Segmentation of HeLa cells in phase-contrast images with and without DAPI stained cell nuclei. Biomed. Technol. 57:519–522, 2012.

    Google Scholar 

  46. 46.

    Wang, Y., Z. Zhang, H. Wang, and S. Bi. Segmentation of the clustered cells with optimized boundary detection in negative phase contrast images. PLoS ONE 10:e0130178, 2015.

    Article  Google Scholar 

  47. 47.

    Witta, S. E., R. M. Gemmill, F. R. Hirsch, C. D. Coldren, K. Hedman, L. Ravdel, B. Helfrich, R. Dziadziuszko, D. C. Chan, M. Sugita, Z. Chan, A. Baron, W. Franklin, H. A. Drabkin, L. Girard, A. F. Gazdar, J. D. Minna, and P. A. Bunn. Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines. Cancer Res. 66:944–950, 2006.

    Article  Google Scholar 

  48. 48.

    Wu, D., X. Ma, and F. Lin. DC electric fields direct breast cancer cell migration, induce EGFR polarization, and increase the intracellular level of calcium ions. Cell Biochem. Biophys. 67:1115–1125, 2013.

    Article  Google Scholar 

  49. 49.

    Yu, Y., C. Feng, Y. Hong, J. Liu, S. Chen, K. M. Ng, K. Q. Luo, and B. Z. Tang. Cytophilic fluorescent bioprobes for long-term cell tracking. Adv. Mater. 23:3298–3302, 2011.

    Article  Google Scholar 

  50. 50.

    Zhao, M. Electrical fields in wound healing-An overriding signal that directs cell migration. Semin. Cell Dev. Biol. 20:674–682, 2009.

    Article  Google Scholar 

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We thank the members of the Asthagiri group for helpful discussions. This work was supported by the National Institutes of Health Grant R01CA138899.

Conflict of Interest

Mark L. Lalli, Brooke Wojeski, and Anand R. Asthagiri declare that they have no conflict of interest.

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No human or animal studies were carried out by the authors for this article.

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Correspondence to Anand R. Asthagiri.

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Associate Editor Michael R. King oversaw the review of this article.

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Lalli, M.L., Wojeski, B. & Asthagiri, A.R. Label-Free Automated Cell Tracking: Analysis of the Role of E-cadherin Expression in Collective Electrotaxis. Cel. Mol. Bioeng. 10, 89–101 (2017).

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  • Cell–cell interactions
  • Electrotaxis
  • Guidance cues
  • Image analysis