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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

The acquisition of invasive behaviour is the key transition in the progression of benign melanocyte hyperplasia to life threatening melanoma. Understanding this transition and the mechanisms of invasion are the key to understanding why malignant melanoma is such a devastating disease and will aid treatment strategies. Underlying the invasive behaviour is increased cell motility caused by changes in cytoskeletal organization and altered contacts with the extra-cellular matrix (ECM). In addition, changes in the interactions of melanoma cells with keratinocytes and fibroblasts enable them to survive and proliferate outside their normal epidermal location. Proteomic and genomic initiatives are greatly increasing our knowledge of which gene products are deregulated in invasive and metastatic melanoma; however, the next challenge is to understand how these genes promote the invasion of melanoma cells. In recent years new models have been developed that more closely recapitulate the conditions of melanoma invasion in vivo. It is hoped that these models will give us a better understanding of how the genes implicated in melanoma progression affect the motility of melanoma cells and their interactions with the ECM, stromal cells and blood vessels. This review will summarise our current understanding of melanoma invasion and focus on the new model systems that can be used to study melanoma.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

The development of malignant melanoma is generally characterized by a series of transitions that are outlined in Figure 1 (Chin, 2003). Melanoma arise from melanocytes that normally reside within the basal layer of the epidermis. Initial aberrant proliferation of melanoyctes gives rise to melanocytic naevi within the epidermis that can show varying degrees of dysplasia. The majority of these do not give rise to malignant disease but a subset will begin to spread (dotted arrow with ‘?’Figure 1). It is also thought than some dysplastic melanocytes do not form naevi but directly form lesions with the capability to spread (curved arrow in Figure 1). The initial spread of dysplastic melanocytes is almost always lateral and within or very near to the epidermis; this is termed the Radial Growth Phase (RGP) and is associated with a good prognosis. However, the next phase, Vertical Growth Phase (VGP), shows pronounced invasion into the dermis and is clinically more dangerous. In the early stages of VGP, the majority of cells remain in clusters with only a few isolated cells breaking away from the bulk of the melanoma (although this is almost impossible to accurately determine from single histological sections). The transition to VGP is associated with the acquisition of metastatic potential as the cells have penetrated the basement membrane and the increasing vascularization of these lesions means that the cells have a readily available route for distant spread. VGP can then progress to more aggressive forms of melanoma that are characterized by extensive vascularization and invasion. In order to understand what causes melanoma invasion it is important to determine the molecular changes that underpin the RGP > VGP transition and the subsequent motility and intravazation of melanoma cells.

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Figure 1.  Development of metastatic melanoma. The progression of a melanocyte to a malignant melanoma is represented. The development of naevi is a relatively frequent event but the transition of naevi to more malignant disease is relatively rare. In addition, malignant disease may arise from melanocytes that do pass through the naevus stage.

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Changes associated with the transition to malignancy

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

Genetic changes

Over the past two decades, numerous causal genetic changes have been identified in melanoma. These include mutations in cell cycle regulators such as CDKN2A/INK4a and various signalling molecules including NRAS, BRAF, PTEN and amplification of AKT3 (Chin, 2003; Davies et al., 2002; Haluska et al., 2006; Omholt et al., 2003; Rodolfo et al., 2004; Sharpless and Chin, 2003). In general the timing of these mutations does not correlate well with the transition from RGP to VGP suggesting that they may not be critical in promoting the invasion of melanoma. This does not exclude a role for these genes in promoting the invasion of melanoma; in particular, there is considerable experimental evidence implicating NRAS, BRAF and PTEN in control of cell motility (this will be discussed in more detail later). Indeed, the situation regarding BRAF may be slightly more complex than first thought; some groups have reported that despite the high prevalence of BRAF mutations in naevi, RGP has a low frequency of mutations that increases upon the transition to VGP (Dong et al., 2003)or that frequency increases as melanoma metastasize (Shinozaki et al., 2004). This apparent contradiction can be partly explained if not all RGP melanoma arise from naevi (Figure 1) or if RGP melanoma arise from BRAF wild-type naevi.

While there are no strong genetic candidates to mediate the switch from RGP to VGP microarray (Bittner et al., 2000; Clark et al., 2000) and immunohistochemical analyses have identified numerous genes whose expression level is altered as melanoma become invasive. Many of these have been subsequently investigated in experimental studies and demonstrated to play a role in melanoma invasion and dissemination.

Changes in gene expression

Some of the changes in gene expression that occur as melanoma develop are important for melanocytes to adapt to the different environment that the dermis presents. For example, reduced E-cadherin levels are likely to reduce interactions with keratinocytes that help to control the behaviour of normal melanocytes (Hsu et al., 1996; Li et al., 2001) and conversely increased N-cadherin may enable melanoma cells to make productive interactions with stromal fibroblasts (Smalley et al., 2005) that may facilitate survival outside the epidermis (Haass et al., 2005; Hsu et al., 2002). The cell adhesion molecule MCAM is also overexpressed as melanoma become more invasive and can promote melanoma cell interactions with endothelial cells and thereby facilitate entry into the vasculature (Bogenrieder and Herlyn, 2002). The expression of many growth factors and cytokines is altered as melanoma become more invasive, including HGF, TGFbeta, FGF and the embryonic morphogen Nodal (al-Alousi et al., 1996; Albino et al., 1991; Topczewska et al., 2006). In addition to autocrine pro-invasive effects on the melanoma cells they can exert paracrine effects that can promote the spread of melanoma. TGF-beta and FGF production can signal to fibroblasts and increase the production of pro-invasive molecules such as tenascin C and HGF (De Wever et al., 2004).

VEGF-A and VEGF-C production by melanoma is crucial for the re-organization and proliferation of endothelial cells leading to the development of both blood and lymphatic vasculature which generates a route for metastatic dissemination. Readers who wish to know more about the complex mechanisms of tumour angiogenesis are directed to the following reviews (Carmeliet and Jain, 2000; Folkman, 2006). While tumour vasculature is usually comprised of endothelial cells it is becoming apparent that melanoma cells can also form part of the vasculature. This process is called vascular mimicry and is thought to be driven by expression of endothelial specific genes in melanoma cells such as ESM-1 and VE-Cadherin (Hendrix et al., 2001; Maniotis et al., 1999). The expression of genes usually associated with other cell types by melanoma may promote their ability to adapt to and move in different environments. Also, reduced expression of genes associated with the normal epidermal environment of melanocytes may aid survival of melanoma cells as they invade. In support of this idea, melanoma cells expressing E-cadherin undergo apoptosis as they start to leave an ‘epidermal’ compartment; whereas cells lacking E-cadherin are able to survive and as a result invade effectively (Hsu et al., 2000). Changes in numerous cell cycle and apoptotic regulators are likely to be crucial for melanoma cells to survive outside their ‘normal’ epidermal environment. Combined gain of Ras and loss of TP53 function can drive invasion and this may be due to increased survival of invasive cells as opposed to a direct effect on cell motility (Chudnovsky et al., 2005).

There are also likely to be numerous translational and post-translational changes associated with the transition to malignancy; it is likely that advances in large scale proteomic analysis will shed light on these in the near future. Changes in the levels of numerous genes implicated in cell motility has been documented in melanoma and in some cases linked to prognosis or metastatic potential. The next section will summarize the changes in cytoskeletal gene expression in melanoma cells and present a generalized mechanism of cell motility.

Changes in cell motility genes in melanoma

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

Cell motility requires the coordination of cell extension, adhesion/de-adhesion and contraction (summarized in Figure 2, also an excellent graphical introduction to cell motility can be found at http://cellix.imba.oeaw.ac.at/). In many cases there are additional ‘sensing’ steps that determine the extent or direction of cell movement and mechanisms, such as proteolysis, to displace the surrounding matrix are required for cells to move within a tissue.

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Figure 2.  Schematic representation of cell motility. The basics individual steps of cell motility are shown together with some of the key regulators that have been functionally implicated in melanoma.

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Extending a protrusion

The first step in cell motility is the extension of a protrusion driven by actin polymerization. The polymerization of actin can be increased by the addition of actin monomers to pre-existing actin filaments or by the Arp2/3 mediated nucleation of new actin filaments (Pollard and Borisy, 2003). The Arp2/3 complex is predominantly regulated by WAVE and WASP family proteins; these are regulated by external stimuli in many ways. Activation of growth factor receptors (TGFalpha, Hepatocyte Growth Factor and SDF-1) will lead to PI-3-K dependent activation the small GTPases Rac1 and Cdc42 which either directly or indirectly form complexes with WAVE and N-WASP (Ridley, 2006; Stradal et al., 2004; Takenawa and Miki, 2001). The importance of these pathways in regulating the actin cytoskeleton, cell motility and metastatic potential of B16 melanoma cells has been shown in a number of studies (Kovacs et al., 2006; Kurisu et al., 2005; Nakagawa et al., 2003; Nakahara et al., 2003). It is also worth noting that these pathways could be triggered by accumulation of PIP3 as a result of mutation or silencing of PTEN which frequently occurs in melanoma (Liliental et al., 2000).

Mechanisms that promote the polymerization of actin on existing filaments have not been studied as extensively in melanoma models at the present time. Nonetheless, it has recently been shown that dephosphorylated cofilin, which generates new barbed ends for actin polymerization by severing existing filaments near the plasma membrane promotes the invasion of melanoma cells (Dang et al., 2006). In breast cancer cells it has been shown that growth factor mediated activation of PLC-gamma can liberate cofilin from binding to PIP2 and thereby increase its ability to generate new actin filaments ends for polymerization (DesMarais et al., 2005; Mouneimne et al., 2004). The actin severing ability of cofilin can be turned off by signalling downstream of Rho family small GTPases, this is likely to be important to ensure that filament severing by cofilin is spatially restricted within the cell and that once actin has been polymerized it is not immediately severed (Kuhn et al., 2000; Maekawa et al., 1999). In other contexts increases in PIP2 levels can promote actin polymerization by binding to and displacing actin filament capping proteins such as gelsolin from the ends of actin filament (Pollard and Borisy, 2003), high levels of gelsolin reduce metastatic spread of melanoma cells (Fujita et al., 2001). All mechanisms of actin polymerization rely on the availability of actin monomers in the cell; these are usually sequestered by molecules such as profilin and thymosin beta4, which is upregulated in metastatic melanoma (Cha et al., 2003). This may help to maintain a larger pool of polymerization competent monomers in invasive melanoma cells and facilitate the formation of actin rich protrusions.

Forming adhesions

Once a cell has extended a protrusion it then needs to form new adhesions to stabilize the protrusion. The major mediators of cell-extracellular matrix are integrins, which form heterodimeric complexes consisting of an alpha and beta sub-unit. Numerous groups have reported deregulated expression of integrins in invasive melanoma and shown the functional importance of integrins (Melchiori et al., 1995; Seftor et al., 1999; Van Belle et al., 1999; reviewed in Kuphal et al., 2005a). The alphaVbeta3 dimer, which binds a range of ligands containing the amino acid sequence RGD (including Fibronectin) appears to be particularly important for the invasive potential of melanoma (Dang et al., 2006; Hsu et al., 1998; Van Belle et al., 1999). The main ECM components that integrins bind are collagen, laminin and fibronectin; peptides that block the ability of integrins to interact with laminin and fibronectin have been shown to reduce melanoma motility in vitro and metastatic spread in vivo (Kuratomi et al., 1999, 2002; Makabe et al., 1990). Non-integrin ECM receptors such as CD44, which binds the common GAG hyaluronic acid, are also upregulated (Hibino et al., 2004; Mummert et al., 2003). The production of matrix components is also altered in invasive melanoma; for example the expression of fibronectin, SPARC, tenascin C and certain laminins is increased in invasive and metastatic melanoma and experimental studies have shown that these matrix components promote melanoma motility (Ilmonen et al., 2004; Ledda et al., 1997; McCarthy and Furcht, 1984; Natali et al., 1995; Pyke et al., 1994). It should be noted that some of these matrix components are produced by stromal fibroblasts associated with the melanoma.

Adhesions need to be connected to the actin cytoskeleton; this is achieved through large multiprotein focal complexes. These may be small and short-lived focal contacts or large and more persistent focal adhesions. In vitro analyses in B16 melanoma cells have demonstrated the sequential recruitment of proteins to newly formed integrin adhesions leading to the development of first focal contacts and subsequently focal adhesions (Ballestrem et al., 2006). The focal complex component alpha-actinin is highly expressed in metastatic melanoma although the function significance of this is unclear (Clark et al., 2000).

Contraction

Once adhesions to the ECM have been formed force needs to be applied to them for the cell body to move. This force is generated by the productive interaction of actin and myosin motor proteins. Acto-myosin interactions are predominantly regulated by phosphorylation in non-muscle cell types and several of the pathways that influence phosphorylation of the regulatory myosin sub-unit are upregulated in metastatic melanoma cells. RhoC is able to promote acto-myosin contractility by binding to ROCK1 and ROCK2 (also called Rho-kinase or ROK alpha) which, in turn, regulate MLC phosphorylation (Fukata et al., 2001). Overexpression of RhoC is a key event in the metastasis of experimental melanoma models and inhibition of RhoC, its close relative RhoA or ROCK 1&2 reduces the invasion of melanoma cells (Clark et al., 2000; Sahai and Marshall, 2003). This is a potential avenue for therapeutic intervention as ROCK kinase inhibitors have been developed that reduce melanoma metastasis in mouse models (Nakajima et al., 2003). The multi-functional protein MTS1 (S100A4) is also deregulated in melanoma and capable of promoting productive acto-myosin interactions (Andersen et al., 2004).

For acto-myosin contraction to effectively move the cell body the F-actin network not only needs to be connected to cell-matrix adhesions but to the plasma membrane. This is achieved by a range of linker proteins which bind F-actin and membrane components such as PIP2 or integral membrane proteins (Bretscher, 1999; Luna, 1991). The expression of one of these proteins, ezrin, increases as melanoma become metastatic (Ilmonen et al., 2005).

De-adhesion

In vitro, most melanoma cells have multiple adhesions at various points around the cell. If contractile force is applied on these adhesions without remodelling of at least some of the adhesions then the cell will generate internal tension but not movement. Therefore detachment of some adhesion complexes is required for effective motility. This has been studied in some detail in vitro and multiple mechanisms of adhesion remodelling have been observed including disassembly, sliding and fragmentation of the cell rear leading to a trail of defunct adhesions behind the cell. The micro-tubule network has been shown to target focal adhesions immediately prior to their disassembly and this may facilitate the dynamin-mediated delivery of proteins that destabilise the adhesions (Ezratty et al., 2005; Kaverina et al., 1999). The kinases FAK and ERK have been shown to promote the turnover of adhesion complexes (Cuevas et al., 2003; Webb et al., 2004). FAK is overexpressed in melanoma (Hess et al., 2005) while ERK1 is overexpressed in one model of metastatic melanoma (Clark et al., 2000) but may be more generally activated downstream of BRAF mutations in melanoma. It is likely that ERK signalling also has a significant function in transcription programmes. Adhesion components can also be cleaved by intra-cellular proteases such as calpain (Franco et al., 2004), which can be regulated by ERK (Glading et al., 2001). Inhibition of calpain can reduce the melanoma migration associated with high levels of integrin alphaVbeta3 (Byzova et al., 2000). In other cases, adhesion complexes may not be disassembled by merely slide behind the cell (Ballestrem et al., 2001). Finally, some melanoma cells may not remodel adhesions at all but instead leave trailing fragments of their cytoplasm (Friedl et al., 1997; Mayer et al., 2004).

Migrating through 3D substrates

The coordinated action of the steps outlined above is capable of producing cell motility in the absence of an ECM barrier. It is possible that cells are able to squeeze through gaps in the ECM or generate sufficient force to deform the ECM and enable movement but these processes will not allow movement through particularly dense ECM. For this to occur proteolysis of ECM components is required. Matrix-MetalloProteinases (MMP) are upregulated in invasive melanoma and there is extensive evidence that they play a role promoting the dissemination of melanoma (Bodey et al., 2001; Hofmann et al., 2005; Hornebeck et al., 2002). However, it is likely that different MMP have pro- and anti-invasion properties and as a result current inhibitors that target broad ranges of MMP have not proved successful in the clinic (Overall and Lopez-Otin, 2002). Another possible reason for the poor results of MMP inhibition in clinical trails is the potential role of non-MMP proteases such as Cathepsins in ECM proteolysis. Cathepsins are frequently deregulated in melanoma and pharmacological inhibition reduces the invasion of melanoma cells (Klose et al., 2006; Mayer et al., 1997). The uPA serine protease and its receptor have also been extensively implicated in melanoma progression possibly through their ability to activate intra-cellular signalling pathways such as the small GTPase Rac1 (see section on extending a protrusion; Kjoller and Hall, 2001).

Co-ordinated regulation of cell motility genes

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

The genes involved in control of cell motility show altered expression levels but not mutational activation in melanoma. This probably reflects the cyclical nature of cell motility processes; if any one of the steps was locked in a hyperactive state independent of the other steps the net result would be very un-coordinated and inefficient motility. Similarly it appears as if there is a coordinated upregulation of genes involved at all the different steps of cell motility. This co-ordinate upregulation of numerous cell motility genes probably reflects the activation of transcriptional programs that promote cell motility by either environmental cues present in invasive melanoma (growth factors or matrix components) or by changes in some master regulatory genes.

In addition to the rapid effects of actin polymerization described above many growth factors promote melanoma migration by increasing the transcription of genes involved in invasion. HGF and TGF-beta are able to upregulate a number of genes involved in melanoma motility; including fibronectin, various collagens, integrin alphaV, CD44 and VEGF-A and downregulate E-cadherin (Berking et al., 2001; Gaggioli et al., 2005; Recio and Merlino, 2003). It is also possible that genetic mutations common in melanoma can promote pro-invasive transcriptional programs in the absence of growth factors. B-raf signalling is capable of promoting Fibronectin expression (Gaggioli et al., 2007) and may also promote the expression of the FN receptor component integrin beta 3 (Woods et al., 2001). One way in which TGF-beta mediates its effects is through the upregulation of the snail family of transcription factors that are capable of driving a mesenchymal pattern of gene expression that is well suited to motility and invasion. Indeed there is much evidence that Snail family proteins are upregulated in invasive melanoma and that this is functionally important for the invasive and metastatic potential of melanoma. Snail proteins are able to decrease E-cadherin, occludin and claudin expression while elevating N-Cadherin levels thereby favouring the interaction of melanoma cells with stromal cells and not keratinocytes (Kajita et al., 2004). Furthermore, Snail co-ordinately regulates the expression of cell motility genes including MMP-2, SPARC, TIMP-1 and RhoA (Kuphal et al., 2005b). The increased activity of Snail family proteins in invasive melanoma probably mimics the activation of these factors during the migration of melanocyte pre-cursor neural crest cells during development. Expression of the Snail-related factor, Slug, in melanocytes has been proposed as a reason why it is easier to generate metastatic tumours from melanocytes than other non-transformed cells using defined genetic lesions (Gupta et al., 2005).

In addition to growth factors triggering co-ordinated pro-invasive changes in gene expression the extra-cellular matrix present in melanoma can promote invasive behaviour. The ECM component SPARC can promote E-cadherin downregulation and Fibronectin upregulation (Robert et al., 2006). Normal melanocytes can be induced to invasive behaviour by exposure to ECM produced by melanoma (Seftor et al., 2005).

Downregulation of other transcriptional programs may also be needed for increased melanoma invasion. AP-2 is downregulated in more aggressive melanoma and forced expression of AP-2 reduces melanoma dissemination (Nyormoi and Bar-Eli, 2003). MITF is a key positive regulator of melanocyte differentiation and it has recently been shown that reduced MITF activity lowers the levels of the actin regulator mDia leading to ROCK-dependent melanoma invasion (Carreira et al., 2006) (see ‘Contraction’ for role of ROCK in melanoma invasion). Interestingly, this was coupled to increased levels of the cell cycle inhibitor p27Kip1; thus transcriptional programs that promote invasion may co-ordinately reduce the proliferation of melanoma cells. While high levels of MITF activity are inconsistent with invasion some levels of MITF activity may be important for maintaining the expression of pro-invasive genes such as c-Met (McGill et al., 2006).

Problems with interpreting results from different systems

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

The model presented thus far seems relatively straight forward with multiple cell motility and invasion genes upregulated in melanoma and either functional or prognostic evidence of their importance in melanoma invasion and progression. On the basis of the evidence presented so far, one might predict that we already know of dozens of molecular targets that when inhibited would block the dissemination of melanoma. Unfortunately, the reality is not so simple; the data has been collated from a very wide range of cell lines, in vitro cell motility assays, in vivo metastasis assays and clinical samples with differing origins and types of analysis. The profound effect of the type of cell motility assay used has been demonstrated by testing the effect of ROCK inhibition on the motility of A375M2 metastatic melanoma cells (Hooper et al., 2006; Sahai and Marshall, 2003). This study showed that depending on the assay used inhibition of ROCK could either modestly promote or dramatically reduce invasion. In particular, when ROCK function was inhibited on rigid 2D substrates there was a slight increase in motility but this was not recapitulated in 3D environments; in fact inhibition of ROCK significantly reduced invasion in this context. When challenged with a deformable 3D barrier Rho and ROCK driven cortical acto-myosin contraction is crucial for pushing the matrix out of the way (Wyckoff et al., 2006) and allowing invasion but on 2D substrates with out a barrier high levels of cortical acto-myosin contraction are not needed.

Although the principles of cell motility outlined above apply to all cells the different steps can be spatially and temporally co-ordinated in very different manners resulting in different ‘modes’ or morphologies of invading cells that show differing degrees of reliance on some of the molecular regulators of cell motility. For example, strong cortical acto-myosin contraction means that A375M2 cells are predominantly rounded when moving in a 3D environment (Sahai and Marshall, 2003). The rapid rounded movement of melanoma cells driven by cortical acto-myosin contraction is generally termed ‘amoeboid’ and has some similarities to the movement of haematopoeitic cells. In contrast WM266.4 cells have lower Rho activity than A375M2 and generally invade with an elongated morphology. These differences in the modes of invasion correspond with a greater dependence of A375M2 on Rho and ROCK function than WM266.4 cells (Sahai and Marshall, 2003). This has dramatic implications for any attempts to translate any in vitro findings regarding melanoma cell line invasion into the clinical situation. The mode of invasion affects the sensitivity of different cell lines to blockade of MMP function; generally cells invading with a rounded morphology characteristic of strong cortical acto-myosin contractility are not sensitive to MMP inhibition because they can use force mediated matrix remodelling in place of proteolysis (Wyckoff et al., 2006). However, cells that do not generate such high levels of force may be more dependent on MMP function. When this was directly tested using WM266.4 cells it was observed that the cells actually converted to a more rounded MMP independent mode of invasion (Sahai and Marshall, 2003). Friedl and colleagues have also demonstrated changes in the mode of tumour cell invasion in response to blockade of either protease or integrin beta 1 function (Hegerfeldt et al., 2002; Wolf et al., 2003). These studies strongly suggest that some melanoma cells can switch their mode of invasion in response to different environmental challenges. The molecular basis of this plasticity is not well understood, microarray analyses have demonstrated that metastatic melanoma cells express genes associated with a diverse range of cell lineages and this may in part explain the diverse modes of motility that melanoma cells can exhibit in vitro (Hendrix et al., 2001). Analysis of clinical samples suggest that melanoma can invade both collectively and as single cells; however, our understanding of switching between modes of invasion and the cytoskeletal regulators critical for the different modes of invasion is still very limited. More ‘realistic’ models of melanoma invasion should help to fill these gaps in our understanding between melanoma cell motility in highly artificial in vitro settings and in patients.

Improved systems for studying melanoma invasion

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

Organotypic cultures

The majority of cell motility assays involve cells moving on rigid two dimensional substrates in the absence of other cell types. While these assays may be easy to perform, they clearly fail to recreate many aspects of the epidermal and dermal environment that invasive melanoma normally encounter. ‘Organotypic’ skin culture systems have been developed that closely recapitulate the epidermal and dermal environments (see Figure 3). When set up with normal keratinocytes the entire differentiation process is established resulting in a ‘tissue’ that has remarkable similarities to normal skin (Mackenzie, 2004). These models can be adapted to include melanocytes or melanoma cells in the keratinocyte layer thereby approximating the physiological situation (Berking and Herlyn, 2001). Invasive melanoma cells will then move into the lower ‘dermal’ layer of the system with pattern of invasion that closely resembles those seen in human melanoma. Similar to human disease, both invasion of clusters of melanoma cells and what appear to be single cells can be observed (Figure 3– it should be noted that the unambiguous identification of isolated invading cells is not possible from single sections). This system has already been used to demonstrate the importance of elevated integrin beta 3 levels in promoting invasion (Hsu et al., 1998), this may be in part through increasing levels of SPARC which may be able to help establish a pro-invasive transcriptional program (Robert et al., 2006; Sturm et al., 2002). Combined activation and inactivation of Ras and TP53 respectively or reduced E-cadherin levels can drive invasion in similar system (Chudnovsky et al., 2005; Hsu et al., 2000); as discussed earlier this may be due the increased ability of invading melanocytes to survive outside the tissue they normally reside in. Histological analysis of these systems suggests that there is a transition from collective invasion to single cell invasion. This will be of particular interest to study because of parallels with the clinical situation and the relative dearth of knowledge about and models for the collective invasion of melanoma cells.

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Figure 3.  Example of an ‘organotypic’ invasive melanoma culture. (A) The experimental set-up is shown. Melanoma cells are seeded within a layer of untransformed keratinocytes and on top of a reconstituted dermal matrix containing fibroblasts. The whole culture in then grown at a gas-liquid interface for 28 days before fixation and analysis. (B) Immuno-staining of ‘organotypic’ invasive melanoma culture. WM266.4 melanoma cells were seeded in a layer of normal HaCat keratinocytes on matrix containing fibroblasts. Paraffin section of the culture after 28 days is shown with WM266.4 cells stained in brown (S100 staining). WM266.4 cells can clearly be seen invading down into the ‘dermal’ layer both in large cluster and potentially as single cells.

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Currently this system has only be used as an end-point assay of melanoma invasion; however, advances in imaging technologies should allow these type of systems to be imaged in real time. This should provide wealth information regarding the cytoskeletal organization of invading melanoma cells and their interactions with keratinocytes and fibroblasts. It should also be possible to introduce other cell types into the system and study the interaction of melanoma with either immune or endothelial cells. There should also be the possibility to use primary cells from melanoma explants in these systems. Indeed, the behaviour of melanoma explants in 3D collagen lattices similar to those used in some organotypic studies has been studied and the requirement for integrin beta 1 for the collective invasion of melanoma demonstrated (Hegerfeldt et al., 2002). Human skin has also been grafted onto immune-compromised mice and melanoma subsequently induced. However, it remains to be seen if these explant systems can be sufficiently manipulated to allow detailed molecular and imaging analyses (Berking and Herlyn, 2001; Chudnovsky et al., 2005). Nonetheless, these explant systems may be suitable for preclinical evaluation of drugs.

Directly imaging melanoma invasion in animal models

The ability to model cancer in the mouse has proved incredibly useful and could potentially give great insights into the mechanisms of melanoma invasion if suitable models were available. The B16 mouse xenograft model of melanoma has been extensively used mainly because it has the benefits of being metastatic and syngeneic with the C57BL6 (Black 6) background. Until recently almost all experiments have relied upon sacrificing animals and then investigating numbers of metastases and/or tumour histology. These methods have the drawback that no dynamic information can be acquired and that cell invasion has to be inferred from histological ‘snap-shots’. However, the development of mouse whole imaging systems (luminescence, fluorescence, MRI, micro-PET) means that the development and spread of a single tumour can be monitored in the same animal over time (Deroose et al., 2007; Hoffman, 2005; Ray et al., 2004). Thus it is now possible to follow the growth and spread of a tumour over a number of days and weeks. The behaviour and motility of single cells can be followed with sub-cellular resolution by live confocal imaging of fluorescently labelled tumours. This can provide highly detailed and direct analysis of melanoma cell motility, interactions with the ECM and other non-tumour cell types. Figure 4 shows a 3D reconstruction of melanoma margin from a live mouse acquired using multiphoton confocal microscopy. A combination of genetically encoded, injectable and intrinsic fluorescent and optical properties have been imaged to reveal tumour cells, blood vessels and collagen fibres. A375 melanoma cells stably expressing GFP are shown in green, tumour vessels are shown in white and the collagen rich matrix surrounding the melanoma is shown in red. The outline of sub-cutaneous fat overlying the tumour can also be seen as slight distortions in the image (marked with dotted line). The movement of tumour cells into the surrounding matrix can be directly visualized by repeated imaging of the same area. One such example is shown in Figure 4B; one A375 cell can be seen moving steadily along a collagen fibre while another moves more rapidly between two fibres and changes direction rapidly. Although this example uses human melanoma cells in an immune-compromised mouse, one benefit of the B16 mouse melanoma model is that cells can be introduced into immunocompetent C57BL6 mice. Further more the effect of the genotype of the non-tumour cells can be studied by introducing B16 cells into genetically modified mice. An elegant and surprising use of this approach was the finding that loss of integrin beta 3 in non-tumour cells actually increased tumour burden through increased tumour angiogenesis (Reynolds et al., 2002).

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Figure 4.  In vivo imaging of invasive melanoma. (A) GFP labelled A375M2 cells were injected sub-cutaneously into immune-compromised mice; once tumour had reached ∼5 mm diameter then mouse was anesthetized, a vascular marker was injected (Angiosense 680 – VISEN) and the overlying skin removed allowing the tumour to be imaged. Collagen fibres are shown in red, A375M2 melanoma cells in green, and vasculature in white. Panel is approx 1 × 1 mm. (B) Images were captured of the same area of tumour repeatedly. A selection of these images is shown with two invading A375M2 melanoma cells. The position of the moving cells in the previous image is shown by the white dotted outline. Panel is approx 300 × 120 μm.

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One major difficulty with the B16 model and other based on cell lines is that cells are injected subcutaneously and this does not accurately reflect the site of origin of melanoma. It is quite likely that by directly injected cells into this site they encounter different ECM compared to melanoma arising in the epidermis and that some of the processes involved in melanoma invasion into the dermis are bypassed. Ideally one would like to have a mouse model of melanoma that arose in situ in the epidermis and then progressed to invasive and malignant behaviour. The development of such a model is complicated by the location of mouse melanocytes in the dermis in neonatal mice and mainly around hair follicles in adult mice. Nonetheless, some models have been developed; HGF overexpression combined with neo-natal UV exposure recapitulates the human disease well and combinations of carcinogens and UV can induce melanoma in some genetic backgrounds (reviewed Noonan et al., 2003; Noonan et al., 2001; Tormo et al., 2006). If these models were crossed with mice expressing suitable fluorescently tagged melanocytes it will be possible to directly image the invasion of melanoma into the dermis and vasculature in living animals. This would be a great step forward in our ability to study the process of melanoma invasion and would open new ways to understand the basic biology of melanoma invasion and to test strategies, either genetic or pharmacological, that aim to block the spread of melanoma. The location of melanoma means that they are easy to access for imaging studies and may therefore serve a paradigm for learning about the invasion of other tumour types.

Models of melanoma also exist in Zebrafish and Xiphophorus that appear to be driven by similar genetic lesions (Dorsky et al., 2000; Patton et al., 2005; Wellbrock et al., 2002). The transparent nature of the fish should facilitate the direct and dynamic imaging of melanoma spread in these models. Recent studies have elegantly demonstrated the high resolution with which cell motility can be studying in the developing zebrafish and have shown that implanted melanoma cells disseminate and promote an angiogenic response in zebrafish (Haldi et al., 2006; Lee et al., 2005).

Concluding remarks

Our understanding of the molecular mechanism of melanoma cell motility has increased greatly in recent years, but this also made us aware of the diverse modes of cell motility and the importance of studying cell motility in appropriate contexts. Exciting times should lie ahead as we begin to explore cell motility in more realistic models of melanoma. In addition, these models will enable detailed molecular and cell biology analysis of the numerous and complex interaction of melanoma cells with other cell types that also contribute to the invasive behaviour of melanoma.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References

We thank Sophie Tartare-Deckert and lab members for their comments, the London Research Institute Histopathology department for technical assistance and Cancer Research UK for financial support.

References

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  2. Summary
  3. Introduction
  4. Changes associated with the transition to malignancy
  5. Changes in cell motility genes in melanoma
  6. Co-ordinated regulation of cell motility genes
  7. Problems with interpreting results from different systems
  8. Improved systems for studying melanoma invasion
  9. Acknowledgements
  10. References
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