Proteolysis-free cell migration through crowded environments via mechanical worrying

Background

Migrating cells underlie multiple key processes in health and disease. Two primary modes of migration have been described: mesenchymal and amoeboid. Mesenchymal migration, a “path-making” mode, is characterized by extracellular matrix (ECM) remodelling via matrix metalloproteinase (MMP) secretion and strong cell-substrate interactions through the formation of focal adhesions. Cells undergoing mesenchymal migration often exhibit an elongated shape with well-organized networks of filamentous actin^1,2.

In contrast, amoeboid migration, a “path-finding” mode, relies on actomyosin contractility and bleb formation with weak cell-substrate adhesions and minimal degradation of the ECM. Instead of carving out paths via MMP secretion, amoeboid cells deform their bodies to fit through pores in the ECM^1-5. Recent studies on melanoma have revealed an enrichment of rounded amoeboid cells at tumour margins with enhanced metastatic capabilities^{6, 7}. Given that most cancer deaths worldwide are associated with metastasis, there is an urgent need to investigate this population of migratory tumour cells.

In this study, Driscoll and colleagues uncover a novel aspect of amoeboid migration wherein cells use polarized blebs to physically degrade ECM fibres in their migratory path. This process of sustained agitation of ECM components is dubbed “worrying.” The authors present a mechanosensitive signalling axis that sustains worrying via adhesion signalling, PI3K/Rac1-mediated actin assembly within large blebs, and matrix degradation that does not rely on MMPs. Importantly, this preprint challenges the pre-existing notion that amoeboid cells do not actively remodel their microenvironment during migration and uses rigorous experimental paradigms to characterize this new type of cell behaviour.

Key Findings

 Amoeboid cells create paths through dense ECM without proteolysis

The authors used metastatic melanoma cells to explore bleb-based motility in soft microenvironments. To mimic dense yet soft in vivo conditions, the authors cultured melanoma cells in fibrous collagen I gels at 1 kPa stiffness. The authors then performed 3D light-sheet microscopy on cells migrating through fluorescently labelled collagen networks and measured the size of pores within the network. They observed that while cells had protrusive blebs that were small enough to fit through most pores, their cell bodies and nuclei were often too large, arguing against a deformation-based mode of migration. Despite their large nuclei and cell bodies, these amoeboid melanoma cells were still able to migrate through collagen networks.

Intriguingly, long-term live-cell imaging revealed the presence of tunnels through the collagen matrix, which contrasts previous claims that amoeboid cells do not degrade the ECM. To determine how amoeboid cells were tunnelling, the authors treated the cells with a general MMP inhibitor. Surprisingly, there was no effect on the melanoma cells’ ability to tunnel. Similar results were obtained when the authors treated the cells with a protease inhibitor cocktail. Together, these results indicate that amoeboid melanoma cells remodel their microenvironment to make migratory paths in a proteolysis-independent manner.

Above: a 3D rendering of a melanoma cell with polarized blebs (coloured by surface curvature) tunnelling through a dense collagen matrix (yellow).

Amoeboid cells make migration paths through dense ECM using large blebs

The authors wondered how amoeboid cells were tunnelling without using proteases. Analysis of live-cell imaging videos revealed that cells within tunnels were highly polarized with respect to bleb position and size, with larger blebs located at the cell front. The authors also noted that collagen fibres were often fragmented in proximity to blebs. Using a 3D optical flow algorithm to analyze the motion of collagen strands near protrusive and retractive blebs on migrating cells, they observed that collagen near the front of highly polarized cells underwent prolonged agitation and internalization via macropinocytosis. Overall, these observations suggest that amoeboid cells use large, polarized blebs to physically ablate collagen fibres in their path.

PI3K activity governs bleb size in migrating cells

The authors sought to uncover molecular players involved in maintaining bleb size and polarity. Staining of the focal adhesion marker paxillin revealed that blebs contacted collagen fibres through adhesion complexes. Strikingly, PI3K, a cell polarity factor recruited by focal adhesion kinase (FAK)^8-10, was also enriched in large blebs at the cell front. Adhesions formed by large blebs were longer-lived than nascent adhesions of cells cultured on coverslips¹¹, suggesting that the location and persistence of adhesions near the migrating cell front recruits PI3K. To confirm this, the authors treated the cells with a small molecule FAK inhibitor. While the bleb number and polarization were unaffected by FAK inhibition, the overall volume of blebs was decreased. Furthermore, when examining the molecular events occurring after FAK inhibition, the authors found that PI3K polarization was abolished followed by reduced bleb volume. To test this causal link directly, they used an optogenetic strategy to enhance PI3K activity with high spatial specificity in migrating cells. Indeed, PI3K photoactivation induced local bleb swelling, whereas treatment with a pharmacological PI3K inhibitor resulted in bleb shrinkage.

PI3K increases bleb size via branched actin polymerization

How does PI3K activity promote bleb growth? Prior studies have suggested that PI3K activity is coupled with actin protrusion, a phenomenon typically associated with mesenchymal rather than amoeboid migration. In contrast, bleb expansion in amoeboid cells is attributed to membrane extrusion driven by cytoplasmic pressure¹². To explore this apparent contradiction, the authors performed total internal reflection fluorescence (TIRF) imaging in live cells expressing beta-actin-GFP and observed that actin filaments assembled inside blebs during expansion. These observations support the hypothesis that bleb growth can also be driven by actin polymerization.

To link PI3K activity with actin filament assembly and bleb growth, the authors investigated the Rac1 – WAVE – Arp2/3 signalling axis, which is active during lamellipodia expansion^13,14. Treating migrating cells with CK666, an Arp2/3 inhibitor, decreased bleb volume. Furthermore, using a light-sensitive Rac1 expression construct, the authors demonstrated that Rac1 photoactivation increased bleb size in a reversible manner. For greater spatial control, they also performed similar experiments using a light-sensitive Tiam1 (a guanine nucleotide exchange factor that specifically acts on Rac1) construct that could be uncaged and sequestered in specific intracellular locations. Consistent with previous results, local Tiam1 activation significantly increased bleb volume, as well as Arp2/3 concentration within the expanding blebs. Altogether, these experiments uncover the connection between PI3K activity and actin-based bleb expansion in “worrying” cells.

Why I chose this preprint

I was highly interested in this preprint because of the characterization of a novel type of cell behaviour during migration. The finding that amoeboid cells physically manipulate and ablate the ECM via mechanical “worrying” challenges the canonical view of amoeboid migration. Further detailed analyses of these behaviours could potentially lead to the development of therapeutic strategies specifically targeting metastatic tumour cells that do not rely on MMPs or other proteases to invade healthy tissue. It would be fascinating to determine whether non-tumoural cell types that undergo amoeboid migration (such as leukocytes) “worry” in normal physiological contexts, or if this behaviour is specific to tumour cells.

In my own research, I am currently investigating a protein that appears to regulate mesenchymal versus amoeboid migration in glioblastoma (GBM), a highly invasive brain tumour. While GBM cells are thought to undergo mesenchymal migration, tissue stiffness within tumours is highly heterogeneous, which may promote different types of migration. It would be interesting to investigate whether invasive GBM cells also exhibit “worrying” behaviour at tumour margins.

 Questions for the authors

1. Aside from PI3K’s well-established role in growth factor signalling that contributes to tumorigenesis, is there any clinical or experimental evidence suggesting that tumours with PI3K overexpression or hyperactivity are more metastatic than tumours with wild type PI3K?
2. Is PI3K activity or expression spatially heterogeneous in solid tumours?
3. In your live-cell imaging experiments, did you observe any cells that exhibited plasticity between the different migration modes (i.e. cells that switched between deformation-based amoeboid migration, “worrying” amoeboid migration, and/or mesenchymal migration)?

References

1. Yamada, K. M., & Sixt, M. (2019). Mechanisms of 3D cell migration. Nature reviews. Molecular cell biology, 20(12), 738–752. https://doi.org/10.1038/s41580-019-0172-9
2. Sahai, E., & Marshall, C. J. (2003). Differing modes of tumour cell invasion have distinct requirements for Rho/ROCK signalling and extracellular proteolysis. Nature cell biology, 5(8), 711–719. https://doi.org/10.1038/ncb1019
3. Ruprecht, V., Wieser, S., Callan-Jones, A., Smutny, M., Morita, H., Sako, K., Barone, V., Ritsch-Marte, M., Sixt, M., Voituriez, R., & Heisenberg, C. P. (2015). Cortical contractility triggers a stochastic switch to fast amoeboid cell motility. Cell, 160(4), 673–685. https://doi.org/10.1016/j.cell.2015.01.008
4. Lämmermann, T., Bader, B. L., Monkley, S. J., Worbs, T., Wedlich-Söldner, R., Hirsch, K., Keller, M., Förster, R., Critchley, D. R., Fässler, R., & Sixt, M. (2008). Rapid leukocyte migration by integrin-independent flowing and squeezing. Nature, 453(7191), 51–55. https://doi.org/10.1038/nature06887
5. Wolf, K., Te Lindert, M., Krause, M., Alexander, S., Te Riet, J., Willis, A. L., Hoffman, R. M., Figdor, C. G., Weiss, S. J., & Friedl, P. (2013). Physical limits of cell migration: control by ECM space and nuclear deformation and tuning by proteolysis and traction force. The Journal of cell biology, 201(7), 1069–1084. https://doi.org/10.1083/jcb.201210152
6. Orgaz, J. L., Crosas-Molist, E., Sadok, A., Perdrix-Rosell, A., Maiques, O., Rodriguez-Hernandez, I., Monger, J., Mele, S., Georgouli, M., Bridgeman, V., Karagiannis, P., Lee, R., Pandya, P., Boehme, L., Wallberg, F., Tape, C., Karagiannis, S. N., Malanchi, I., & Sanz-Moreno, V. (2020). Myosin II Reactivation and Cytoskeletal Remodeling as a Hallmark and a Vulnerability in Melanoma Therapy Resistance. Cancer cell, 37(1), 85–103.e9. https://doi.org/10.1016/j.ccell.2019.12.003
7. Georgouli, M., Herraiz, C., Crosas-Molist, E., Fanshawe, B., Maiques, O., Perdrix, A., Pandya, P., Rodriguez-Hernandez, I., Ilieva, K. M., Cantelli, G., Karagiannis, P., Mele, S., Lam, H., Josephs, D. H., Matias-Guiu, X., Marti, R. M., Nestle, F. O., Orgaz, J. L., Malanchi, I., Fruhwirth, G. O., … Sanz-Moreno, V. (2019). Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment. Cell, 176(4), 757–774.e23. https://doi.org/10.1016/j.cell.2018.12.038
8. Chen, H. C., & Guan, J. L. (1994). Association of focal adhesion kinase with its potential substrate phosphatidylinositol 3-kinase. Proceedings of the National Academy of Sciences of the United States of America, 91(21), 10148–10152. https://doi.org/10.1073/pnas.91.21.10148
9. Johnson, H. E., King, S. J., Asokan, S. B., Rotty, J. D., Bear, J. E., & Haugh, J. M. (2015). F-actin bundles direct the initiation and orientation of lamellipodia through adhesion-based signaling. The Journal of cell biology, 208(4), 443–455. https://doi.org/10.1083/jcb.201406102
10. Welf, E. S., Ahmed, S., Johnson, H. E., Melvin, A. T., & Haugh, J. M. (2012). Migrating fibroblasts reorient directionality by a metastable, PI3K-dependent mechanism. The Journal of cell biology, 197(1), 105–114. https://doi.org/10.1083/jcb.201108152
11. Choi, C. K., Vicente-Manzanares, M., Zareno, J., Whitmore, L. A., Mogilner, A., & Horwitz, A. R. (2008). Actin and alpha-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nature cell biology, 10(9), 1039–1050. https://doi.org/10.1038/ncb1763
12. Cunningham C. C. (1995). Actin polymerization and intracellular solvent flow in cell surface blebbing. The Journal of cell biology, 129(6), 1589–1599. https://doi.org/10.1083/jcb.129.6.1589
13. Welch, H. C., Coadwell, W. J., Stephens, L. R., & Hawkins, P. T. (2003). Phosphoinositide 3-kinase-dependent activation of Rac. FEBS letters, 546(1), 93–97. https://doi.org/10.1016/s0014-5793(03)00454-x
14. Bisi, S., Disanza, A., Malinverno, C., Frittoli, E., Palamidessi, A., & Scita, G. (2013). Membrane and actin dynamics interplay at lamellipodia leading edge. Current opinion in cell biology, 25(5), 565–573. https://doi.org/10.1016/j.ceb.2013.04.001

Tags: #amoeboid, cell migration, extracellular matrix, melanoma

doi: https://doi.org/10.1242/prelights.33209

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