Keeping in touch with contact inhibition of locomotion

1In multicellular organisms, cell migration is essential for normal development and is required throughout life for numerous processes, including wound healing and responses to infections. Disregulation in the control of cell migration can lead to, or exacerbate, human diseases such as cancer, atherosclerosis and chronic inflammatory pathologies. More than a century of research in this area has generated a detailed morphological description of moving cells and this has allowed researchers to deepen their understanding of the molecular mechanisms that control cell polarity and cell protrusions during migration. An important concept to emerge is the idea that most cells do not move as isolated entities in vivo but rather interact with their neighbours during migration. Even cells of the immune system that can migrate singly have to interact with other non-motile cells along their migratory paths. Thus, cells must have their locomotory machinery adapted to these constant interactions. This has prompted scientists for decades to try to investigate the ‘social behaviour of cells’. However, how cells interact during migration is still not fully understood.

2More than five decades ago, Abercrombie and Heaysman found that the direction of migration of fibroblasts cultured in vitro was affected by their interaction with other cells. They called this process ‘contact inhibition of locomotion’ (CIL, see Refs., Box 1) and it was proposed as an explanation for wound healing of epithelia, as this inhibition of cell contact dependent cell migration was released during wound healing, allowing the migration of the cells at the border of the wound. The potential importance of this idea became immediately apparent when they observed that malignant mesenchymal cells showed a reduced CIL response, being able to invade fibroblast cultures in what was compared to invasive metastasis (see Refs ). Nonetheless, several factors led to a gradual loss of interest in the basis of this phenomenon. The molecular mechanism that orchestrates CIL has remained elusive for decades with only few recent advances. This is partly owing to the fact that evidence for CIL occurring in vivo has been sparse. Moreover, a different process, involving cell division rather than locomotion, was also named contact inhibition, leading to some confusion in the literature (see Glossary). Finally, there are also some common misconceptions, for example that CIL only happens when cells collide, or that its sole function is to inhibit migration.

3In this article, we revisit the data that indicate CIL is a crucial mechanism for cell migration in vivo. Recent advances support a view of CIL as a general mechanism of local inhibition of cell protrusions in migratory cells. This leads to directional migration via the redirection of colliding single cells. Also, it is possible that CIL leads to directional movement in collective cell migration via promotion of coherence among cells. Thus, we propose that CIL could play an important role in coordinating the migration of this recognized mode of migration in embryonic and cancer cells. Ongoing improvements in live imaging allow us to analyze CIL in vivo to test its importance in this context. We will review the few molecules reported to be involved in CIL. Moreover, recent advances in our understanding of the molecular bases of cell migration will allow us to propose more candidate molecules that mediate CIL and a molecular link among them.

4The concept of CIL describes the observed behaviour of a cell to change the direction of its movement after contact with another cell (Box 1). The typical sequence of cell activities implicated in CIL are: (i) cell–cell contact, (ii) inhibition of cell protrusive activities at the site of contact, (iii) generation of a new protrusion away from the site of cell contact and (iv) migration in the direction of the new protrusion (Figure 1a). However, this sequence can be modified by different factors. For example, one of the cells might not be responsive to the other and thus, only one of the cells will be redirected. The number of surrounding cells can also alter the outcome of CIL. This four-step sequence is usually observed when individual cells, such as two fibroblasts, collide. However, in a sheet of cells only the cells at the free edge will produce lamellipodia whereas cells in contact with others at the centre of the cluster will generate smaller and more transient protrusions, if any. In this case, CIL will lead to the inhibition of cell protrusions of the inner cells in a cluster (Figure 1b). If a cluster of packed cells has a free edge, only the cells at the leading edge will produce protrusions. This can lead to directional migration of the whole cluster (Figure 1b),. As a consequence of this behaviour, cells exhibiting CIL do not crawl over their neighbours leading to monolayer formation in groups and to scattering in single cells.

5The lamellipodium has been described as the typical locomotory apparatus used to sense the adjacent cells during CIL; however, it is possible that other cell protrusions are also involved in this phenomenon (Box 2). For example, it has been recently proposed that filopodia could be the actual sensory structure in CIL. This could mediate a type of CIL that would operate at longer distances than the cell body size and would probably involve collapse of protrusions but not necessarily a contact between the cell bodies. In fact, analysis of neural crest (NC) migration in vivo shows that these cells establish filopodia-like contacts with neighbouring cells and that this contact is sufficient to promote CIL.

6Spatial cues such as chemoattractants are usually considered to explain the persistent orientation of cell polarity that leads to directional migration. CIL can also contribute to cell polarization because the cells form their protrusions away from the cell–cell contact. Molecules localized to the cell–cell contact will be absent from the leading edge and therefore contribute to polarity. Thus, CIL does not only halt migration of cells but also allow cells to re-polarize and migrate in a new direction, serving as another type of spatial cue. This is crucial during the migration of cells that disperse from an original common location, which is a frequent feature found in embryo development. For example, endodermal cells from mouse and zebrafish embryos are initially localized in a specific region of the forming body from where they disperse to colonize their final destinations (respiratory and digestive tracts), or the myeloid cells, formed in the ventral region of the Xenopus embryo that need to scatter along the entire epidermis. When migration of all these kinds of cells is carefully analysed, a clear suggestion of CIL behaviour is observed (refer to the supplementary videos in the aforementioned references). Cell collisions lead to a change in the direction of migration, upon which cells move away from each other. In addition, these cells rarely overlap and when protrusions are visible, they seem to retract upon contact. These features suggest that these migratory cells, and probably many others, exhibit CIL in vivo. The observation that cells are constantly contacting each other argues against the role of a chemorepellent as a mechanism of dispersion; although it is probable that a combination of different mechanisms drives cell migration in this and other processes during development.

7Several mechanisms are known to operate during cell migration in vivo such as random walk, chemoattraction and cell intercalation. However, time lapse analysis of these migrating cells is consistent with CIL being an additional mechanism that contributes to cell migration. For example, the migration of Cajal-Retzius cells, a transient neuronal population crucial for the development of the brain cortex, is controlled by the apposed meningeal membranes, which produce and secrete the chemokine Cxcl12. However, this chemokine seems to be uniformly distributed along the migratory space and what provides the directionality in the dispersion of these cells are ‘contact-inhibitory interactions’, which correspond to CIL. Indeed the assay used to characterize the interactions between these cells is the same as the one used by Abercrombie and Heaysman when CIL was initially described. These observations suggest that CIL could be a general migratory mechanism that co-exists with processes such as chemoattraction, random-walk and cell intercalation to re-set the polarity of migratory cells.

8In multicellular organisms, cells often move in groups rather than as singular cells. Cell migration in loosely or closely associated groups has been called collective cell migration (reviewed in Refs ). Collective cell migration is now a widely recognized mode of migration during embryogenesis and cancer. Both collective cell migrations and CIL are defined by the ability of cells to interact with their neighbours during migration and it is probable that these two processes are linked. There is a wide variety of collective cell migration, from sheets of migrating cells found in carcinomas and in head mesoderm of amphibian embryos (Figure 2a, ), to closely associated clusters of cells such as the migration of the lateral line in zebrafish, border cells in Drosophila embryos or melanomas (Figure 2b, ). Other cells are organized in chains such as Drosophila myoblasts or squamous cell carcinoma (Figure 2c, ). Another example of this is the migration of endothelial cells during sprouting in angiogenesis, in which inhibition of cell protrusion between the cells and presence of large lamellipodia and filopodia in the leader cells has been compared to CIL. Another mode of collective cell migration has been called streaming (Figure 2d), and has been found in the migration of neural crest cells, mammalian endoderm and possibly in some breast carcinomas. In this type of migration, the cells move as a loose cluster in which individual cells can be identified but are constantly interacting with each other. Interestingly, it has been shown that during neural crest migration, an example of cell streaming, cells make local and transient contacts which are required for CIL.

9Despite the diversity of types of collective migration, there is a common theme for all of them: they all have major protrusions at the leading edge and show a high degree of organization and coordination during migration, which are features of CIL. Although the degree of inhibition of cell protrusions between cells is variable and in some cases cryptic protrusions are observed between cells, it is tempting to speculate that the inhibition of protrusions in cell clusters during collective cell migration is based on CIL. If so, the two types of CIL – in single cell migration (Figure 1a) and in collective movements (Figure 1b) – would represent two aspects of the same process. It has been shown that CIL plays a role in collective migration of mesenchymal cells, such as neural crest, but there is no evidence that it could have a similar role in migration of more epithelial cells. Further knowledge of the molecular basis of CIL is required to compare it with the molecular mechanism that inhibits cell protrusions during collective cell migration.

10The molecular mechanisms that regulate CIL are still unknown, which is largely owing to the lack of investigation and debate on this topic. Despite the sparse data available, it is still possible to dissect CIL into two core cellular mechanisms requiring two different types of molecular machineries. First, cells need to sense the contact with other cells. This mechanism needs to be mediated by molecules located at the cell surface and to have a cognate ligand/receptor pair on the surface of the contacting cell. Moreover, molecules mediating the contact are also required to be able to transduce the signal from the juxtaposed cell into the responding cell. This response is the second mechanism. Upon contact, cells require a mechanism that regulates the withdrawal of protrusions at the contact region followed by the formation of a new protrusion elsewhere. Thus, the second mechanism is basically a repolarization mechanism. Importantly, molecules involved in these two mechanisms have been described as required for proper CIL. However, these two mechanisms have not been directly linked to each other. In this section we will review this evidence and propose a possible molecular link between these findings. Also we propose other surface molecules that could be mediating CIL (Box 3).

11Different pieces of information suggest that molecules, usually linked with cell–cell adhesion, are likely to mediate CIL. Although at a first sight there might be an apparent contradiction between CIL and adhesion, there is actually a long-standing link between these two mechanisms. Although CIL implies cell repulsion and dispersion, it also requires adhesion to strengthen the contact and to allow cell–cell signalling to occur. In fact, the establishment of transient adhesion points between colliding cells has been observed in vitro before their lamellipodia are retracted owing to CIL. Moreover, it is clear that adhesion molecules do not only provide mechanical adhesion but they also work as ligand/receptor pairs playing an important role in cell signalling.

12One of these molecule families is cadherins. Cadherins are a multigene family of cell surface glycoproteins that mediate Ca2+-dependent homophilic cell–cell adhesion by their extracellular domains. Moreover, they can activate intracellular signals involved in cell polarity and in cytoskeleton control such as RhoA and Ena/VASP. Cadherins were among the first molecules that were directly implicated in CIL. E-cadherin has been shown to be required for CIL, and interestingly not for contact inhibition of proliferation (CIP), in migratory cells and in confluent epithelial cells. Likewise, the XB/U-cadherin in Xenopus (similar to the mammalian P-cadherin) is required for the contact-dependent coordination in collective migration of head mesoderm. Interestingly, a recent similar study in fish embryos has shown the requirement of E-cadherin for collective migration of mesoderm in vivo. Although the authors mostly attribute its role to cell–cell adhesion, they also suggest a possible role for CIL in mesoderm migration. An appealing hypothesis would be that both adhesion and CIL converge at the level of E-cadherin to control collective migration. This is supported by the role of E-cadherin in CIL and the link between CIL and adhesion. Altogether, these data indicate that cadherins are essential for CIL. At the same time they highlight that different tissues or cell types could use different cadherins or even other adhesion molecules during CIL (Box 3).

13A key step in CIL is the inhibition of cell protrusions and the re-setting of the intracellular polarity. Cell protrusions are dynamic and complex structures that are formed largely by actin filaments and are regulated by intricate molecular networks (see Box 2, Ref. ). Thus, the inhibition of cell protrusions is not a passive mechanism but, instead, requires the activation of a complex regulatory machinery. Members of the family of small Rho GTPases are essential in the control of both cell polarity and protrusions (Box 2). RhoA is known to control myosin II-dependent contraction of the trailing end of a cell through the protein kinase ROCK. Therefore, RhoA can be a negative regulator of protrusion formation and, thus, a good candidate for a protrusion inhibitory mechanism. The spatial and temporal control of RhoA activity is crucial for appropriate inhibition of cell protrusions. This control can be exerted by a variety of molecules, among which, we find cell adhesion molecules and members of the non-canonical Wnt or planar cell polarity (PCP) signalling pathway.

14Recent evidence in neural crest cells shows that RhoA is involved in CIL via the PCP pathway. Here it was shown that CIL members of the PCP signalling would be activated at cell–cell contacts, which in turn locally activate RhoA. Activated RhoA would then antagonise Rac1 and inhibit cell protrusions. Interestingly, a similar mechanism has been described for the inhibition of cell protrusions by cell–cell contact during vasculogenesis. Although this is an interesting mechanism, it does not fully account for how the presence of an adjacent cell is transduced into this intracellular mechanism. However, with the literature available it is possible to propose a molecular link between cadherins and RhoGTPases.

15Activation of different Rho GTPases appears to be cell type and cadherin-type dependent. It is well established that cadherin engagement leads to activation of Rac1 and Cdc42 and inhibition of RhoA at the cell contact region of many cells. However, activation of RhoA as a result of cell–cell adhesion has been reported in keratinocytes and in N-cadherin dependent cell–cell adhesion of C2C12 myoblasts. It has also been shown that association of N-cadherin with p120 (a catenin that binds to the intracellular domain of cadherins) in cholesterol-rich microdomains leads to activation of RhoA during myogenesis. Whether a similar mechanism occurs in migratory cells remains unknown. It is known that one of the functions of p120ctn is the regulation of Rho GTPases, which has led to suggest that the inhibitory activity of p120ctn on RhoA is dependent on the cytoplasm localisation of p120ctn. Expression of different cadherins sequestrates p120ctn to the membrane and blocks the inhibition of RhoA by p120ctn, suggesting that formation of cadherin-based cell–cell adhesion established during CIL sequesters p120ctn to the membrane and away from cytoplasmic pools therefore relieving the inhibition of RhoA activity (Refs ). In addition, it has been shown that inhibition of sprouting during vasculogenesis requires VE-cadherin, which in turn activates RhoA and inhibits Rac1 at cell junctions, in a process reminiscent of CIL.

16It is probable that different cells use different molecules to interact with their neighbours, giving more versatility to CIL. This greater flexibility could explain why the same cell can exhibit CIL with one particular kind of cell but not with others. Other molecules such as Ephrins/Eph and Notch/Delta and PCP proteins are also possible mediators of CIL (Box 3).

17The first crucial contribution of CIL to cancer research was the idea that cell locomotion is a normal activity of somatic cells that needs to be restricted for the cells to remain at the right place within an organism. This restriction comes from neighbouring cells, which when absent, will allow the migration of cells liberated from this repression. Malignancy, thus, is not necessarily the acquisition of motility by cancerous cells but the absence or lessening of the response to the inhibition of migration exerted by neighbours. This intrinsic but inhibited tendency to migrate of at least some somatic cells has been proposed as important for normal physiological processes such as wound healing of epithelia. In addition to CIL the activity of growth factors also plays a role to stimulate re-epithelization and to produce significant changes in adhesion and cell morphology during wound healing.

18To better understand the effect of CIL in normal and cancerous cells, it is necessary to keep in mind that there are two types of CIL. When two cells (or groups of cells) of the same type encounter each other they can exhibit CIL or not. If they do, it is said that they have homotypic CIL (see Glossary). This has been extensively documented in chick heart fibroblast, among other cell types. Similarly, two different cell types can encounter each other and also display CIL, which would then correspond to heterotypic CIL (see Glossary; Figure 3a). Chick heart fibroblasts, for example, exhibit this behaviour when confronted with normal mouse muscle fibroblasts.

19By contrast, several sarcoma and melanoma cell lines have diminished or absent CIL: they will invade territories populated by other cells, such as normal fibroblasts (Figure 3b). This led to the conclusion that absence of CIL between tumour and normal cells was at the basis of invasive metastasis. A common misunderstanding of these observations is to believe that malignant cells have lost CIL between themselves. It has been shown that malignant cells have lost heterotypic CIL when confronted with normal fibroblasts but they usually do not lose homotypic CIL (Ref., P. Friedl, personal communication). This is similar to what happens to neural crest cells that have homotypic CIL (among neural crest cells) but can invade mesoderm and other tissues during their migration. An appealing hypothesis would be that the invasive behaviour of tumours is facilitated by the absence of heterotypic CIL with normal cells, whereas homotypic CIL between cancer cells helps collective migration and/or dispersion of the tumour. Also, it should be noted that CIL in malignant cells is not always lost but sometimes diminished. For example, the invasion of S180 sarcoma cells is almost completely unobstructed by chick heart fibroblasts, whereas that of mouse melanomas or BAS56 sarcoma cells is partially blocked. The implications of CIL in the invasive properties of tumours in vivo remain to be studied. A recent study showed that the activation of the cytoplasmic form of the human oncogene MET in mouse liver progenitor cells (MLP29) produced loss of contact inhibition. Interestingly, when these cells are transplanted into the spleen of immunodeficient mice they become highly invasive carcinomas.

20The usual misconception that cancer cells have lost contact inhibition only holds true for CIP (see Glossary) and not for CIL between cancer cells. It has been proposed that CIP and CIL are molecularly similar, but the evidence for this is scarce, and the simple observation that many tumours do not have CIP but retain CIL with other cancer cells, strongly suggests that these are two different processes. The molecular basis of CIL in cancer is a fascinating, but poorly developed, area of research that could have important practical implications. Understanding the molecular mechanism by which cancer cells have lost CIL with neighbouring cells could lead to new diagnostic and therapeutic tools.

21Collective migration is now a well-recognized mechanism of migration both in morphogenesis and in cancer progression. Thus, the cellular and molecular understanding of the cell–cell interactions in this type of migration is crucial. CIL is a cellular interaction likely to be crucial for collective migration. However, a better description of its molecular basis in vivo is needed, as well as an understanding as to whether CIL is actually linked to collective cell migration (see Box 4 for outstanding questions about CIL).

22At the same time development of fluorescent transgenic animals and improvements in microscopy techniques, such as confocal microscopy and time lapse imaging, will help to accelerate our molecular description of CIL in vivo. These techniques provide non-invasive imaging tools that allow us to investigate cell migration in vivo in genetically modifiable organisms where the role of different molecules can be tested.

23These are early days in understanding the molecular basis of CIL. However, we have dissected it into two sequential mechanisms with different molecular players. They involve a cell adhesion molecule-dependent first step in which the colliding cells sense each other. This mechanism is followed by a repolarization of the cell. We provide evidence favouring a role for cadherins in the first process, followed by a RhoGTPases-dependent repolarization during the second. Also, we have proposed a link between these two processes. However, other molecules could also be mediating CIL and further investigation is needed to establish a detailed molecular explanation of CIL and to elucidate its cell-type specificity. This is a crucial step because it would allow us to investigate the problem of why most cancerous cell can reduce the heterotypic CIL while maintaining homotypic CIL among them.

24The role of CIL in collective migration also raises an interesting problem of how large groups of cells can self-organize from local cell–cell interactions. Self-organization of migratory groups of cells have been described in several morphogenetic movements such as the lateral line and the migration of tumour cohorts. Thus, CIL together with other local interactions such as cell–cell adhesion could organise tissues at a much larger scale. In fact, recent mathematical models have successfully reproduced the behaviour of migratory sheets by only considering local interactions at the single cell level, especially CIL. These cell–cell interactions can complement other mechanisms, such as taxis and physical forces, in promoting migration of groups and clusters of cells. The molecular characterization of CIL is just starting to be unravelled but the increasing evidence of its importance will promote a better understanding of this process in normal and pathological cell migration.