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Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma

A Publisher Correction to this article was published on 27 September 2022

This article has been updated


During wound repair, branching morphogenesis and carcinoma dissemination, cellular rearrangements are fostered by a solid-to-liquid transition, known as unjamming. The biomolecular machinery behind unjamming and its pathophysiological relevance remain, however, unclear. Here, we study unjamming in a variety of normal and tumorigenic epithelial two-dimensional (2D) and 3D collectives. Biologically, the increased level of the small GTPase RAB5A sparks unjamming by promoting non-clathrin-dependent internalization of epidermal growth factor receptor that leads to hyperactivation of the kinase ERK1/2 and phosphorylation of the actin nucleator WAVE2. This cascade triggers collective motility effects with striking biophysical consequences. Specifically, unjamming in tumour spheroids is accompanied by persistent and coordinated rotations that progressively remodel the extracellular matrix, while simultaneously fluidizing cells at the periphery. This concurrent action results in collective invasion, supporting the concept that the endo-ERK1/2 pathway is a physicochemical switch to initiate collective invasion and dissemination of otherwise jammed carcinoma.

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Fig. 1: Endocytic reawakening of motility is dependent on EGFR activation.
Fig. 2: RAB5A increases non-clathrin internalization of EGFR.
Fig. 3: RAB5A endosomal ERK1/2 activity is required for flocking locomotion.
Fig. 4: RAB5A overcomes kinetic and proliferation arrest in terminally differentiated acini.
Fig. 5: RAB5A promotes the emergence of coordinated angular rotation in cancer spheroids.
Fig. 6: RAB5A promotes collective invasion in tumour spheroids and tumour slices.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its supplementary Information files and from the corresponding authors upon reasonable request.

Code availability

The codes used for the analysis are all indicated in the Methods.

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  1. Hakim, V. & Silberzan, P. Collective cell migration: a physics perspective. Rep. Prog. Phys. 80, 076601 (2017).

    Article  Google Scholar 

  2. Haeger, A., Wolf, K., Zegers, M. M. & Friedl, P. Collective cell migration: guidance principles and hierarchies. Trends Cell Biol. 25, 556–566 (2015).

    Article  Google Scholar 

  3. Park, J. A., Atia, L., Mitchel, J. A., Fredberg, J. J. & Butler, J. P. Collective migration and cell jamming in asthma, cancer and development. J. Cell Sci. 129, 3375–3383 (2016).

    CAS  Google Scholar 

  4. Bi, D., Yang, X., Marchetti, M. C. & Manning, M. L. Motility-driven glass and jamming transitions in biological tissues. Phys. Rev. X 6, 021011 (2016).

    Google Scholar 

  5. Sadati, M., Taheri Qazvini, N., Krishnan, R., Park, C. Y. & Fredberg, J. J. Collective migration and cell jamming. Differentiation 86, 121–125 (2013).

    Article  CAS  Google Scholar 

  6. Atia, L. et al. Geometric constraints during epithelial jamming. Nat. Phys. 14, 613–620 (2018).

    Article  CAS  Google Scholar 

  7. Garcia, S. et al. Physics of active jamming during collective cellular motion in a monolayer. Proc. Natl Acad. Sci. USA 112, 15314–15319 (2015).

    Article  CAS  Google Scholar 

  8. Oswald, L., Grosser, S., Smith, D. M. & Kas, J. A. Jamming transitions in cancer. J. Phys. D 50, 483001 (2017).

    Article  Google Scholar 

  9. Haeger, A., Krause, M., Wolf, K. & Friedl, P. Cell jamming: collective invasion of mesenchymal tumor cells imposed by tissue confinement. Biochim. Biophys. Acta 1840, 2386–2395 (2014).

    Article  CAS  Google Scholar 

  10. Sigismund, S. & Scita, G. The ‘endocytic matrix reloaded’ and its impact on the plasticity of migratory strategies. Curr. Opin. Cell Biol. 54, 9–17 (2018).

    Article  CAS  Google Scholar 

  11. Corallino, S., Malabarba, M. G., Zobel, M., Di Fiore, P. P. & Scita, G. Epithelial-to-mesenchymal plasticity harnesses endocytic circuitries. Front. Oncol. 5, 45 (2015).

    Article  Google Scholar 

  12. Palamidessi, A. et al. Endocytic trafficking of Rac is required for the spatial restriction of signaling in cell migration. Cell 134, 135–147 (2008).

    Article  CAS  Google Scholar 

  13. Frittoli, E. et al. A RAB5/RAB4 recycling circuitry induces a proteolytic invasive program and promotes tumor dissemination. J. Cell Biol. 206, 307–328 (2014).

    Article  CAS  Google Scholar 

  14. Malinverno, C. et al. Endocytic reawakening of motility in jammed epithelia. Nat. Mater. 16, 587–596 (2017).

    Article  CAS  Google Scholar 

  15. Giavazzi, F. et al. Giant fluctuations and structural effects in a flocking epithelium. J. Phys. D 50, 384003 (2017).

    Article  Google Scholar 

  16. Giavazzi, F. et al. Flocking transitions in confluent tissues. Soft Matter 14, 3471–3477 (2018).

    Article  CAS  Google Scholar 

  17. Mendoza, M. C. Phosphoregulation of the WAVE regulatory complex and signal integration. Semin. Cell Dev. Biol. 24, 272–279 (2013).

    Article  CAS  Google Scholar 

  18. Debnath, J., Muthuswamy, S. K. & Brugge, J. S. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods 30, 256–268 (2003).

    Article  CAS  Google Scholar 

  19. Levitzki, A. & Gazit, A. Tyrosine kinase inhibition: an approach to drug development. Science 267, 1782–1788 (1995).

    Article  CAS  Google Scholar 

  20. Lang, E. et al. Coordinated collective migration and asymmetric cell division in confluent human keratinocytes without wounding. Nat. Commun. 9, 3665 (2018).

    Article  Google Scholar 

  21. Zeigerer, A. et al. Rab5 is necessary for the biogenesis of the endolysosomal system in vivo. Nature 485, 465–470 (2012).

    Article  CAS  Google Scholar 

  22. Kirchhausen, T., Owen, D. & Harrison, S. C. Molecular structure, function, and dynamics of clathrin-mediated membrane traffic. Cold Spring Harbor Perspect. Biol. 6, a016725 (2014).

    Article  Google Scholar 

  23. Sigismund, S. et al. Clathrin-mediated internalization is essential for sustained EGFR signaling but dispensable for degradation. Dev. Cell 15, 209–219 (2008).

    Article  CAS  Google Scholar 

  24. Johannes, L., Parton, R. G., Bassereau, P. & Mayor, S. Building endocytic pits without clathrin. Nat. Rev. Mol. Cell. Biol. 16, 311–321 (2015).

    Article  CAS  Google Scholar 

  25. Caldieri, G. et al. Reticulon 3-dependent ER-PM contact sites control EGFR nonclathrin endocytosis. Science 356, 617–624 (2017).

    Article  CAS  Google Scholar 

  26. Koivusalo, M. et al. Amiloride inhibits macropinocytosis by lowering submembranous pH and preventing Rac1 and Cdc42 signaling. J. Cell Biol. 188, 547–563 (2010).

    Article  CAS  Google Scholar 

  27. Di Guglielmo, G. M., Baass, P. C., Ou, W. J., Posner, B. I. & Bergeron, J. J. Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma. EMBO J. 13, 4269–4277 (1994).

    Article  Google Scholar 

  28. Vieira, A. V., Lamaze, C. & Schmid, S. L. Control of EGF receptor signaling by clathrin-mediated endocytosis. Science 274, 2086–2089 (1996).

    Article  CAS  Google Scholar 

  29. McDonald, P. H. et al. Beta-arrestin 2: a receptor-regulated MAPK scaffold for the activation of JNK3. Science 290, 1574–1577 (2000).

    Article  CAS  Google Scholar 

  30. Villaseñor, R., Nonaka, H., Del Conte-Zerial, P., Kalaidzidis, Y. & Zerial, M. Regulation of EGFR signal transduction by analogue-to-digital conversion in endosomes. eLife 4, e06156 (2015).

    Article  Google Scholar 

  31. Barrett, S. D. et al. The discovery of the benzhydroxamate MEK inhibitors CI-1040 and PD 0325901. Bioorg. Med. Chem. Lett. 18, 6501–6504 (2008).

    Article  CAS  Google Scholar 

  32. Kirchhausen, T., Macia, E. & Pelish, H. E. Use of dynasore, the small molecule inhibitor of dynamin, in the regulation of endocytosis. Methods Enzymol. 438, 77–93 (2008).

    Article  CAS  Google Scholar 

  33. Komatsu, N. et al. Development of an optimized backbone of FRET biosensors for kinases and GTPases. Mol. Biol. Cell 22, 4647–4656 (2011).

    Article  CAS  Google Scholar 

  34. Itoh, F. et al. The FYVE domain in Smad anchor for receptor activation (SARA) is sufficient for localization of SARA in early endosomes and regulates TGF-β/Smad signalling. Genes Cells 7, 321–331 (2002).

    Article  CAS  Google Scholar 

  35. Farooqui, R. & Fenteany, G. Multiple rows of cells behind an epithelial wound edge extend cryptic lamellipodia to collectively drive cell-sheet movement. J. Cell Sci. 118, 51–63 (2005).

    Article  CAS  Google Scholar 

  36. Alekhina, O., Burstein, E. & Billadeau, D. D. Cellular functions of WASP family proteins at a glance. J. Cell Sci. 130, 2235–2241 (2017).

    CAS  Google Scholar 

  37. Mendoza, M. C. et al. ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex. Mol. Cell 41, 661–671 (2011).

    Article  CAS  Google Scholar 

  38. Innocenti, M. et al. Abi1 is essential for the formation and activation of a WAVE2 signalling complex. Nat. Cell Biol. 6, 319–327 (2004).

    Article  CAS  Google Scholar 

  39. Steffen, A. et al. Sra-1 and Nap1 link Rac to actin assembly driving lamellipodia formation. EMBO J. 23, 749–759 (2004).

    Article  CAS  Google Scholar 

  40. Ewald, A. J., Brenot, A., Duong, M., Chan, B. S. & Werb, Z. Collective epithelial migration and cell rearrangements drive mammary branching morphogenesis. Dev. Cell 14, 570–581 (2008).

    Article  CAS  Google Scholar 

  41. Kim, H. Y. & Nelson, C. M. Extracellular matrix and cytoskeletal dynamics during branching morphogenesis. Organogenesis 8, 56–64 (2012).

    Article  Google Scholar 

  42. Carey, S. P., Martin, K. E. & Reinhart-King, C. A. Three-dimensional collagen matrix induces a mechanosensitive invasive epithelial phenotype. Sci. Rep. 7, 42088 (2017).

    Article  CAS  Google Scholar 

  43. Nguyen-Ngoc, K. V. et al. ECM microenvironment regulates collective migration and local dissemination in normal and malignant mammary epithelium. Proc. Natl Acad. Sci. USA 109, E2595–E2604 (2012).

    Article  CAS  Google Scholar 

  44. Miller, F. R., Santner, S. J., Tait, L. & Dawson, P. J. xenograft model of human comedo ductal carcinoma in situ. J. Natl Cancer Inst. 92, 1185–1186 (2000).

    Article  CAS  Google Scholar 

  45. Dvorak, H. F. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N. Engl. J. Med. 315, 1650–1659 (1986).

    Article  CAS  Google Scholar 

  46. Vader, D., Kabla, A., Weitz, D. & Mahadevan, L. Strain-induced alignment in collagen gels. PLoS ONE 4, e5902 (2009).

    Article  Google Scholar 

  47. Storm, C., Pastore, J. J., MacKintosh, F. C., Lubensky, T. C. & Janmey, P. A. Nonlinear elasticity in biological gels. Nature 435, 191–194 (2005).

    Article  CAS  Google Scholar 

  48. Munster, S. et al. Strain history dependence of the nonlinear stress response of fibrin and collagen networks. Proc. Natl Acad. Sci. USA 110, 12197–12202 (2013).

    Article  CAS  Google Scholar 

  49. Staneva, R., Barbazan, J., Simon, A., Vignjevic, D. M. & Krndija, D. Cell migration in tissues: explant culture and live imaging. Methods Mol. Biol. 1749, 163–173 (2018).

    Article  CAS  Google Scholar 

  50. Yang, P. S. et al. Rab5A is associated with axillary lymph node metastasis in breast cancer patients. Cancer Sci. 102, 2172–2178 (2011).

    Article  CAS  Google Scholar 

  51. Das, T. et al. A molecular mechanotransduction pathway regulates collective migration of epithelial cells. Nat. Cell Biol. 17, 276–287 (2015).

    Article  CAS  Google Scholar 

  52. Mongera, A. et al. A fluid-to-solid jamming transition underlies vertebrate body axis elongation. Nature 561, 401–405 (2018).

    Article  CAS  Google Scholar 

  53. Dang, T. T., Esparza, M. A., Maine, E. A., Westcott, J. M. & Pearson, G. W. ΔNp63α promotes breast cancer cell motility through the selective activation of components of the epithelial-to-mesenchymal transition program. Cancer Res. 75, 3925–3935 (2015).

    Article  CAS  Google Scholar 

  54. Innocenti, M. et al. Abi1 regulates the activity of N-WASP and WAVE in distinct actin-based processes. Nat. Cell Biol. 7, 969–976 (2005).

    Article  CAS  Google Scholar 

  55. Stradal, T. E. et al. Regulation of actin dynamics by WASP and WAVE family proteins. Trends Cell Biol. 14, 303–311 (2004).

    Article  CAS  Google Scholar 

  56. Barry, D. J., Durkin, C. H., Abella, J. V. & Way, M. Open source software for quantification of cell migration, protrusions, and fluorescence intensities. J. Cell Biol. 209, 163–180 (2015).

    Article  CAS  Google Scholar 

  57. Kardash, E., Bandemer, J. & Raz, E. Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat. Protoc. 6, 1835–1846 (2011).

    Article  CAS  Google Scholar 

  58. Yang, Y. L., Leone, L. M. & Kaufman, L. J. Elastic moduli of collagen gels can be predicted from two-dimensional confocal microscopy. Biophys. J. 97, 2051–2060 (2009).

    Article  CAS  Google Scholar 

  59. Beznoussenko, G. V., Ragnini-Wilson, A., Wilson, C. & Mironov, A. A. Three-dimensional and immune electron microscopic analysis of the secretory pathway in Saccharomyces cerevisiae. Histochem. Cell Biol. 146, 515–527 (2016).

    Article  CAS  Google Scholar 

  60. Beznoussenko, G. V. & Mironov, A. A. Correlative video-light-electron microscopy of mobile organelles. Methods Mol. Biol. 1270, 321–346 (2015).

    Article  CAS  Google Scholar 

  61. Park, J. A. et al. Unjamming and cell shape in the asthmatic airway epithelium. Nat. Mater. 14, 1040–1048 (2015).

    Article  CAS  Google Scholar 

  62. Pastore, R., Pesce, G. & Caggioni, M. Differential variance analysis: a direct method to quantify and visualize dynamic heterogeneities. Sci. Rep. 7, 43496 (2017).

    Article  CAS  Google Scholar 

  63. Barron, J. L., Fleet, D. J. & Beauchemin, S. S. Performance of optical flow techniques. Int. J. Comput. Vis. 12, 43–77 (1994).

    Article  Google Scholar 

  64. Kubo, R. The fluctuation-dissipation theorem. Rep. Prog. Phys. 29, 255–284 (1966).

    Article  CAS  Google Scholar 

  65. Castro, A. P. et al. Combined numerical and experimental biomechanical characterization of soft collagen hydrogel substrate. J. Mater. Sci. Mater. Med. 27, 79 (2016).

    Article  CAS  Google Scholar 

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This work has been supported by: the Associazione Italiana per la Ricerca sul Cancro (AIRC) to G.S. (IG#18621), P.P.D.F (IG#18988 and MCO 10.000), and F.G. (MFAG#22083); the Italian Ministry of University and Scientific Research (MIUR) to P.P.D.F. and G.S. (PRIN: PROGETTI DI RICERCA DI RILEVANTE INTERESSE NAZIONALE – Bando 2017#2017HWTP2K); the Italian Ministry of Health (RF-2013-02358446) to G.S. Regione Lombardia and CARIPLO foundation (Project 2016-0998) to R.C.; Worldwide Cancer Research (WCR#16-1245) to S.S. C.M. and F.G. are partially supported by fellowships from the University of Milan, E.B. from the FIRC-AIRC. We thank J. Christian (Max Planck Institute for Medical Research, Heidelberg, Germany) for help with fluorescent beads.

Author information

Authors and Affiliations



A.P., C.M. and E.F. designed and performed all the experiments and edited the manuscript. S.C. aided in generating cell lines and in the analysis of immunofluorescence and kinematic studies. E.B., S.S. and P.P.F.D. conceived the internalization assays and interpreted the trafficking results. G.V.B. performed EM studies. E.M., M.G. and D.P. aided in all the imaging acquisition, FRET and PIV analysis. C.T aided in the analysis of RAB5A expression in breast cancer. Q.L. and F.A. performed and analysed the AFM measurements. F.G. and R.C. analysed all the kinematic data, developed the tools for 3D motility and mechanical analysis, edited the manuscript and conceived part of the study together with C.M. E.A.C.-A helped in setting up the fluorescent bead assay. G.S. conceived the whole study, wrote the manuscript and supervised all the work. C.M., F.G., R.C. and G.S. are all equally responsible for this work.

Corresponding authors

Correspondence to Chiara Malinverno, Fabio Giavazzi, Roberto Cerbino or Giorgio Scita.

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The authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–12, Supplementary Tables 1–4, Supplementary Video Legends 1–29, Supplementary Discussion and Supplementary references 1–19

Reporting Summary

Supplementary Video 1

RAB5A reawakening of collective motion in jammed epithelial monolayers depends on EGF

Supplementary Video 2

RAB5A reawakening of collective motion in jammed epithelial monolayers depends on EGFR

Supplementary Video 3

EGF-dependence of RAB5A flocking motility in EGFP-H2B-jammed epithelia monolayers

Supplementary Video 4

PIV analysis of EGF and EGFR-dependent endocytic unjamming

Supplementary Video5

RAB5A flocking motion in jammed epithelia is reduced by silencing Dynamin 2

Supplementary Video 6

RAB5A flocking motion in jammed epithelia is reduced by silencing RTN3, but not RTN4

Supplementary Video 7

RAB5A, but neither RAB5B nor RAB5C induces flocking motion in jammed epithelia

Supplementary Video 8

Flocking motion is abrogated by treatment with inhibitors of the MAPK/ERK1/2 pathway

Supplementary Video 9

Flocking motion is abrogated by treatment with Dynasore

Supplementary Video 10

MEK-DD is not sufficient to reawaken collective motion in jammed MCF10A monolayers

Supplementary Video 11

RAB5A-induced cryptic lamellipodia is compromised by treatment with MEK1/2 inhibitor

Supplementary Video 12

RAB5A-induced cryptic lamellipodia are inhibited by silencing the WAVE complex

Supplementary Video 13

RAB5A reawakening of collective motion in jammed epithelia is impaired by silencing NAP1

Supplementary Video 14

Silencing of NAP1 or WAVE2 affects RAB5A-induced wound closure in epithelial monolayers

Supplementary Video 15

Acini kinematic motility assay

Supplementary Video 16

PIV analysis on acini motility

Supplementary Video 17

RAB5A-mediated unjamming in MCF10A acini is trafficking-, EGFR- and ERK1/2-dependent

Supplementary Video 18

RAB5A-induced angular motion of MCF10A is independent of cell proliferation

Supplementary Video 19

RAB5A overcomes kinetic arrest of differentiated MCF10A acini

Supplementary Video 20

RAB5A reawakens collective motion in jammed carcinoma cells

Supplementary Video 21

RAB5A promotes wound closure and flocking motion

Supplementary Video 22

RAB5A-mediated unjamming induces coordinated angular rotation in breast cancer spheroids

Supplementary Video 23

3D DVA of a RAB5A rotating spheroid after removal of the global rotation

Supplementary Video 24

RAB5A-flocking in spheroids is trafficking-, EGFR-, ERK1/2- and ARP2/3-dependent

Supplementary Video 25

RAB5A-mediated 3D unjamming promotes collective invasion in tumour spheroids

Supplementary Video 26

RAB5A spheroids exert larger stresses on surrounding ECM

Supplementary Video 27

Instantaneous velocity and stress maps of control and RAB5A-expressing spheroids.

Supplementary Video 28

RAB5A-mediated flocking promotes collective invasion in ex vivo DCIS tumour slices

Supplementary Video 29

PIV analysis on ex vivo DCIS tumour slice motility

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Palamidessi, A., Malinverno, C., Frittoli, E. et al. Unjamming overcomes kinetic and proliferation arrest in terminally differentiated cells and promotes collective motility of carcinoma. Nat. Mater. 18, 1252–1263 (2019).

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