Observation and analysis of cancer cell behaviour in 3D environment is essential for full understanding of the mechanisms of cancer cell invasion. However, label-free imaging of live cells in 3D conditions is optically more challenging than in 2D. Quantitative phase imaging provided by coherence controlled holographic microscopy produces images with enhanced information compared to ordinary light microscopy and, due to inherent coherence gate effect, enables observation of live cancer cells' activity even in scattering milieu such as the 3D collagen matrix. Exploiting the dynamic phase differences method, we for the first time describe dynamics of differences in cell mass distribution in 3D migrating mesenchymal and amoeboid cancer cells, and also demonstrate that certain features are shared by both invasion modes. We found that amoeboid fibrosarcoma cells' membrane blebbing is enhanced upon constriction and is also occasionally present in mesenchymally invading cells around constricted nuclei. Further, we demonstrate that both leading protrusions and leading pseudopods of invading fibrosarcoma cells are defined by higher cell mass density. In addition, we directly document bundling of collagen fibres by protrusions of mesenchymal fibrosarcoma cells. Thus, such a non-invasive microscopy offers a novel insight into cellular events during 3D invasion.

Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University Průmyslová 595 252 42 Vestec u Prahy Czech Republic

Central European Institute of Technology Brno University of Technology Purkyňova 656 123 612 00 Brno Czech Republic

Department of Cell Biology Charles University Viničná 7 Prague Czech Republic

Institute of Physical Engineering Faculty of Mechanical Engineering Brno University of Technology Technická 2896 2 Brno 616 00 Czech Republic

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Sleeman J, Steeg PS. Cancer metastasis as a therapeutic target. Eur. J. Cancer. 2010;46:1177–1180. PubMed PMC

Lazebnik Y. What are the hallmarks of cancer? Nat. Rev. Cancer. 2010;10:232–233. PubMed

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

Wolf K, et al. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J. Cell Biol. 2003;160:267–277. PubMed PMC

Rösel D, et al. Up-regulation of Rho/ROCK signaling in sarcoma cells drives invasion and increased generation of protrusive forces. Mol. Cancer Res. 2008;6:1410–20. PubMed

Lämmermann T, Sixt M. Mechanical modes of ‘amoeboid’ cell migration. Curr. Opin. Cell Biol. 2009;21:636–44. PubMed

Charras GT, Coughlin M, Mitchison TJ, Mahadevan L. Life and times of a cellular bleb. Biophys. J. 2008;94:1836–53. PubMed PMC

Panková K, Rösel D, Novotný M, Brábek J. The molecular mechanisms of transition between mesenchymal and amoeboid invasiveness in tumor cells. Cell. Mol. Life Sci. 2010;67:63–71. PubMed PMC

Friedl P, Wolf K. Tumour-cell invasion and migration: diversity and escape mechanisms. Nat. Rev. Cancer. 2003;3:362–74. PubMed

Clark AG, Vignjevic DM. Modes of cancer cell invasion and the role of the microenvironment. Curr. Opin. Cell Biol. 2015;36:13–22. PubMed

Friedl P. Prespecification and plasticity: shifting mechanisms of cell migration. Curr. Opin. Cell Biol. 2004;16:14–23. PubMed

Micuda S, Rosel D, Ryska A, Brabek J. ROCK inhibitors as emerging therapeutic candidates for sarcomas. Curr. Cancer Drug Targets. 2010;10:127–134. PubMed

Rosel D, Brabek J, Vesely P, Fernandes M. Drugs for solid cancer: the productivity crisis prompts a rethink. Onco. Targets. Ther. 2013;6:767–777. PubMed PMC

Gandalovičová A, et al. Migrastatics—Anti-metastatic and Anti-invasionDrugs: Promises and Challenges. Trends in Cancer. 2017;3:391–406. PubMed PMC

Edmondson R, Broglie JJ, Adcock AF, Yang L. Three-Dimensional Cell Culture Systems and Their Applications in Drug Discovery and Cell-Based Biosensors. Assay Drug Dev. Technol. 2014;12:207–218. PubMed PMC

Tolde O, Rösel D, Veselý P, Folk P, Brábek J. The structure of invadopodia in a complex 3D environment. Eur. J. Cell Biol. 2010;89:674–80. PubMed

Tolde O, Rosel D, Janostiak R, Vesely P, Brabek J. Dynamics and morphology of focal adhesions in complex 3D environment. Folia Biol. (Praha). 2012;58:177–184. PubMed

Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science. 2001;294:1708–1712. PubMed

Leith EN, et al. Imaging through scattering media using spatial incoherence techniques. Opt. Lett. 1991;16:1820–1822. PubMed

Lostak M, Chmelik R, Slaba M, Slaby T. Coherence-controlled holographic microscopy in diffuse media. Opt. Express. 2014;22:4180–4195. PubMed

Kollarova V, Collakova J, Dostal Z, Vesely P, Chmelik R. Quantitative phase imaging through scattering media by means of coherence-controlled holographic microscope. J. Biomed. Opt. 2015;20:111206. PubMed

Chmelik R, et al. The Role of Coherence in Image Formation in Holographic Microscopy. Prog. Opt. 2014;59:267–335.

Pastorek L, Venit T, Hozak P. Holography microscopy as an artifact-free alternative to phase-contrast. Histochem. Cell Biol. 2018;149:179–186. PubMed

Davies HG, Wilkins MHF. Interference Microscopy and Mass Determination. Nature. 1952;169:541. PubMed

Barer R. Interference microscopy and mass determination. Nature. 1952;169:366–367. PubMed

Zangle TA, Teitell MA. Live-cell mass profiling: an emerging approach in quantitative biophysics. Nat. Methods. 2014;11:1221–1228. PubMed PMC

Taddei ML, et al. Mesenchymal to amoeboid transition is associated with stem-like features of melanoma cells. Cell Commun. Signal. 2014;12:24. PubMed PMC

MacKay JL, Kumar S. Simultaneous and independent tuning of RhoA and Rac1 activity with orthogonally inducible promoters. Integr. Biol. (Camb). 2014;6:885–894. PubMed PMC

Rodriguez-Hernandez, I., Cantelli, G., Bruce, F. & Sanz-Moreno, V. Rho, ROCK and actomyosin contractility in metastasis as drug targets. F1000Research5, F1000 Faculty Rev-783 (2016). PubMed PMC

Wyckoff JB, Pinner SE, Gschmeissner S, Condeelis JS, Sahai E. ROCK- and myosin-dependent matrix deformation enables protease-independent tumor-cell invasion in vivo. Curr. Biol. 2006;16:1515–23. PubMed

Wolf K, et al. Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion. Nat Cell Biol. 2007;9:893–904. PubMed

van Helvert S, Friedl P. Strain Stiffening of Fibrillar Collagen during Individual and Collective Cell Migration Identified by AFM Nanoindentation. ACS Appl. Mater. Interfaces. 2016;8:21946–21955. PubMed

Krizova A, et al. Dynamic phase differences based on quantitative phase imaging for the objective evaluation of cell behavior. J. Biomed. Opt. 2015;20:111214. PubMed

Petrie RJ, Yamada KM. Multiple mechanisms of 3D migration: the origins of plasticity. Curr. Opin. Cell Biol. 2016;42:7–12. PubMed PMC

Friedl P, Wolf K. Plasticity of cell migration: a multiscale tuning model. J. Cell Biol. 2010;188:11–9. PubMed PMC

Wolf K, et al. Physical limits of cell migration: Control by ECM space and nuclear deformation and tuning by proteolysis and traction force. J. Cell Biol. 2013;201:1069–1084. PubMed PMC

McGregor AL, Hsia C-R, Lammerding J. Squish and squeeze—the nucleus as a physical barrier during migration in confined environments. Curr. Opin. Cell Biol. 2016;40:32–40. PubMed PMC

Wang W, et al. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res. 2002;62:6278–6288. PubMed

Condeelis J, Segall JE. Intravital imaging of cell movement in tumours. Nat. Rev. Cancer. 2003;3:921–930. PubMed

Egeblad M, Rasch MG, Weaver VM. Dynamic interplay between the collagen scaffold and tumor evolution. Curr. Opin. Cell Biol. 2010;22:697–706. PubMed PMC

Elkhatib, N. et al. Tubular clathrin/AP-2 lattices pinch collagen fibers to support 3D cell migration. Science356 (2017). PubMed

Petrie RJ, Yamada KM. At the leading edge of three-dimensional cell migration. J. Cell Sci. 2012;125:5917–5926. PubMed PMC

Paul NR, et al. alpha5beta1 integrin recycling promotes Arp2/3-independent cancer cell invasion via the formin FHOD3. J. Cell Biol. 2015;210:1013–1031. PubMed PMC

Doyle AD, Carvajal N, Jin A, Matsumoto K, Yamada KM. Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions. Nat. Commun. 2015;6:8720. PubMed PMC

Chhabra ES, Higgs HN. The many faces of actin: matching assembly factors with cellular structures. Nat. Cell Biol. 2007;9:1110–1121. PubMed

Galbraith CG, Yamada KM, Galbraith JA. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science. 2007;315:992–995. PubMed

Schafer C, et al. One step ahead: role of filopodia in adhesion formation during cell migration of keratinocytes. Exp. Cell Res. 2009;315:1212–1224. PubMed

Lämmermann T, et al. Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature. 2008;453:51–5. PubMed

Schmidt S, Friedl P. Interstitial cell migration: integrin-dependent and alternative adhesion mechanisms. Cell Tissue Res. 2010;339:83–92. PubMed PMC

Paňková D, et al. NG2-mediated Rho activation promotes amoeboid invasiveness of cancer cells. Eur. J. Cell Biol. 2012;91:969–977. PubMed

Friedl P, Borgmann S, Brocker EB. Amoeboid leukocyte crawling through extracellular matrix: lessons from the Dictyostelium paradigm of cell movement. J. Leukoc. Biol. 2001;70:491–509. PubMed

Ma M, Baumgartner M. Filopodia and membrane blebs drive efficient matrix invasion of macrophages transformed by the intracellular parasite Theileria annulata. Plos One. 2013;8:e75577. PubMed PMC

Park K, et al. Measurement of adherent cell mass and growth. Proc. Natl. Acad. Sci. USA. 2010;107:20691–20696. PubMed PMC

Cermak N, et al. High-throughput measurement of single-cell growth rates using serial microfluidic mass sensor arrays. Nat. Biotechnol. 2016;34:1052–1059. PubMed PMC

Martinez-Martin D, et al. Inertial picobalance reveals fast mass fluctuations in mammalian cells. Nature. 2017;550:500–505. PubMed

Lee K, et al. Quantitative Phase Imaging Techniques for the Study of Cell Pathophysiology: From Principles to Applications. Sensors (Basel). 2013;13:4170–4191. PubMed PMC

Janeckova H, Vesely P, Chmelik R. Proving tumour cells by acute nutritional/energy deprivation as a survival threat: a task for microscopy. Anticancer Res. 2009;29:2339–2345. PubMed

Balvan J, et al. Multimodal holographic microscopy: distinction between apoptosis and oncosis. Plos One. 2015;10:e0121674. PubMed PMC

Collakova J, et al. Coherence-controlled holographic microscopy enabled recognition of necrosis as the mechanism of cancer cells death after exposure to cytopathic turbid emulsion. J. Biomed. Opt. 2015;20:111213. PubMed

Balvan J, et al. Oxidative Stress Resistance in Metastatic Prostate Cancer: Renewal by Self-Eating. Plos One. 2015;10:e0145016. PubMed PMC

Gal B, et al. Distinctive behaviour of live biopsy-derived carcinoma cells unveiled using coherence-controlled holographic microscopy. Plos One. 2017;12:e0183399. PubMed PMC

Calin VL, et al. Evaluation of the metastatic potential of malignant cells by image processing of digital holographic microscopy data. FEBS Open Bio. 2017;7:1527–1538. PubMed PMC

Driscoll MK, Danuser G. Quantifying modes of 3D cell migration. Trends Cell Biol. 2015;25:749–759. PubMed PMC

Strbkova L, Zicha D, Vesely P, Chmelik R. Automated classification of cell morphology by coherence-controlled holographic microscopy. J. Biomed. Opt. 2017;22:1–9. PubMed

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