The high mortality of melanoma is caused by rapid spread of cancer cells, which occurs unusually early in tumour evolution. Unlike most solid tumours, thickness rather than cytological markers or differentiation is the best guide to metastatic potential. Multiple stimuli that drive melanoma cell migration have been described, but it is not clear which are responsible for invasion, nor if chemotactic gradients exist in real tumours. In a chamber-based assay for melanoma dispersal, we find that cells migrate efficiently away from one another, even in initially homogeneous medium. This dispersal is driven by positive chemotaxis rather than chemorepulsion or contact inhibition. The principal chemoattractant, unexpectedly active across all tumour stages, is the lipid agonist lysophosphatidic acid (LPA) acting through the LPA receptor LPAR1. LPA induces chemotaxis of remarkable accuracy, and is both necessary and sufficient for chemotaxis and invasion in 2-D and 3-D assays. Growth factors, often described as tumour attractants, cause negligible chemotaxis themselves, but potentiate chemotaxis to LPA. Cells rapidly break down LPA present at substantial levels in culture medium and normal skin to generate outward-facing gradients. We measure LPA gradients across the margins of melanomas in vivo, confirming the physiological importance of our results. We conclude that LPA chemotaxis provides a strong drive for melanoma cells to invade outwards. Cells create their own gradients by acting as a sink, breaking down locally present LPA, and thus forming a gradient that is low in the tumour and high in the surrounding areas. The key step is not acquisition of sensitivity to the chemoattractant, but rather the tumour growing to break down enough LPA to form a gradient. Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

Melanoma is feared because it spreads very rapidly when tumours are relatively small. It is not known why this metastasis is so efficient and aggressive. In particular, it is not known what drives melanoma cells to start to migrate out from the tumour. Here, we have studied the chemical signals that guide the migration of melanoma cells. We find that a component of serum, lysophosphatidic acid (LPA), functions as a remarkably strong attractant for all of the melanoma cells that we examined. We also observe that melanoma cells rapidly break down LPA. We conclude that melanomas create their own gradients of LPA, with low LPA in the tumour and high LPA outside. Since melanoma cells are attracted by LPA, this LPA gradient around the melanomas serves as a signal that drives the tumour cells out into the surrounding skin and blood vessels. Finally, we show that such gradients exist in a mouse model of melanoma. Self-generated LPA gradients are therefore an intriguing new driver for melanoma dispersal.

Funding: This research was funded by Cancer Research UK core grants to RI, LM, and OS; by the Wellcome Trust (fellowship 095186/Z/10/Z to AMM and programme grant to DB); and by the BBSRC (core grant to MJOW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Muinonen-Martin et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

If cells that are responding to a stimulus are also responsible for breaking it down, the result is a self-generated gradient. Under these conditions the gradient is always oriented away from the current location of the cells. One such example has been shown during the development of the zebrafish lateral line primordium [19] – [21] , in which a dummy receptor locally absorbs an SDF-1 stimulus to set up a gradient that is detected by a different receptor. In this work we find that melanoma cells self-generate chemotactic gradients from unlocalised, exogenous LPA. These gradients tend to direct cells to disperse outwards from tumours, thus directly promoting metastasis. Furthermore, we measure LPA gradients across real melanomas in vivo. Since melanomas of sufficient size both generate their own LPA gradients and respond to them, chemotaxis-steered spread of melanomas is almost inevitable.

Alternatively, local gradients may be formed from signals that are widely produced, but are absorbed or broken down locally. This local depletion mechanism is potentially just as effective as local production, but less often invoked. In the cancer literature, only localised sources are typically invoked, for example individual macrophages within the vasculature attracting cancer cells within the tumour [10] .

The suggested role of chemoattractants in cancer dispersal—whether growth factors, chemokines, or LPA—raises the crucial question of how gradients are generated. Chemotaxis will only work with signals that are presented as gradients—homogeneous signals contain no directional information—and the steeper the gradient, the more efficient the chemotaxis. Chemical gradients are typically effective over distances of less than a millimetre—limits on the efficiency of diffusion make larger gradients impractical [18] . Thus for a gradient to be formed there must be a gradient source that is close to the tumour.

Chemotaxis assays are typically performed in transwell chambers, in which cells are grown on one side of a membrane filter and potential attractants are added to the other side. Chemotaxis is assayed by the number of cells observed on the far side of the filter after a fixed interval. These assays are subject to a wide range of artifacts. Cells' behaviour during chemotaxis cannot be studied, which makes it extremely difficult to distinguish chemotaxis from directionless changes in migratory behaviour (i.e., chemokinesis [14] ). Potential attractants form extremely steep and rather short-lived concentration gradients, unlike the physiological conditions the assay aims to reproduce. More seriously still, conditions either side of the filter may be discretely different; cells may grow, survive, or adhere better on one side of the filter than the other, giving changes in the numbers of cells that can be artifactually interpreted as chemotaxis. Direct viewing chambers, such as Dunn, Zigmond, or Insall chambers, are more laborious to use but yield a far higher quality of data, with fewer artifacts [15] – [17] . In work described here, we use direct-viewing chambers to identify lysophosphatidic acid (LPA) as a far more potent chemoattractant for melanoma cells than other previously described attractants. We have developed and refined two direct-viewing assays to assess mechanisms of cell dispersal and chemotaxis, allowing us to distinguish chemotactic from chemokinetic and contact-driven responses under defined conditions that minimize artifacts. Furthermore, the use of direct-viewing chambers makes comparison of attractants' relative efficiencies practical.

In the melanoma literature, most chemotaxis is attributed to growth factors such as platelet-derived growth factor (PDGF) and EGF [11] and the CXCR4 ligand SDF-1 [12] , though a wide variety of potential attractants have been discussed [13] . Gradients of growth factor or SDF-1 have not been identified in vivo, they can only be inferred from the cells' behaviour or pattern of responses in vitro.

(A) Schematic showing the stages of melanoma spread. (B) WM239A metastatic melanoma cells dispersing in uniform medium. 2×10 4 cells were introduced into one reservoir of an Insall chamber containing complete medium with 10% FBS throughout, and observed by time-lapse phase contrast microscopy. See Movie S1 . The left side of each image shows the reservoir containing cells, while the right side is the viewing bridge of the chamber. (C–D) Migration is density-dependent. WM1158 metastatic melanoma cells were seeded at different densities in full medium with 10% FBS, and observed as before. At 2×10 4 cells/well and above, peak migration distances increase sharply, as confirmed by the distance at 17 hours (D; graph shows mean ± SEM). (E) Migration is not driven by production of a repellent. 2×10 4 WM1158 cells were introduced into a chamber in minimal medium without serum and observed at 17 hours as before. Cells survive and adhere, but do not disperse. (F) Migration is not driven by production of a serum-derived repellent. 2×10 4 WM1158 cells were introduced into a chamber in minimal medium without serum and observed at 17 hours as before. Cells disperse less efficiently in conditioned medium than in fresh medium. (G) Migration mediated by chemotaxis up a serum gradient is similar to density-induced migration. Left panel: 2×10 4 WM1158 cells were introduced into a chamber in the presence of a gradient from 0% FBS around the cells to 10% in the opposite reservoir [15] . The cells rapidly migrate towards the well containing serum. Right panel: similar assay with 10% serum in both reservoirs. Panels taken from Movies S3 and S1 , respectively.

However, several questions about melanoma progression remain unanswered. The first is what drives melanomas to change from the relatively benign radial growth phase (RGP) to the far more invasive vertical growth phase (VGP) (see schematic diagram in Figure 1A ). In RGP melanomas, cells only spread horizontally along the basement membrane, compared to VGP melanoma cells, which are also capable of spreading both upwards into the epidermis (Pagetoid spread) and downwards, into and through the dermis (invasion). This spread raises the related question, of what drives cells to migrate away from the primary tumour. Simple, random migration is an extremely inefficient way of dispersing cells and also unlikely to drive cells to invade through matrix and basement membranes. Chemotaxis—cell migration directed by gradients of soluble signalling molecules—is implicated as an important driver of metastasis by a wide range of data [7] , [8] , and is considered necessary to drive efficient invasion. In breast cancer, for example, some tumour cells migrate towards epidermal growth factor (EGF) [9] . However, EGF gradients have only been inferred in vivo, never measured, and their sources are usually unclear. In the case of breast cancer, the EGF is thought to be secreted by macrophages recruited in a paracrine loop by the tumour [10] , but for other attractants and cell types the sources of chemotactic signals are not known.

One principal reason behind the aggressiveness of melanoma derives from the developmental history of melanocytes, the pigment producing cells in the skin that mutate to form melanomas. During mammalian development melanoblasts, the melanocyte precursors, emerge from a restricted location at the neural crest, and migrate rapidly from there throughout the developing dermis, before maturing into melanocytes on the basement membrane of the epidermis [6] . Thus a substantial level of cell migration is required for even skin pigmentation. Even in adults—for example following treatment for vitiligo—melanocytes can spread significant distances from the hair follicles to repopulate the surrounding skin. The melanocyte lineage is thus inherently migratory.

Melanoma is an unusually aggressive cancer, which often metastasizes early during tumour development [1] . Tumours that have not clinically metastasized are frequently curable, but patients are far less likely to survive if tumours have metastasized before they are surgically removed, and metastasis is the principal cause of cancer mortality [2] . The most influential prognostic factor in predicting metastasis and survival is the thickness of the tumour (the “Breslow depth”) [3] . There is a dramatic increase in the risk of metastasis with only millimeter increases in Breslow depth [3] . This characteristic is unlike most solid tumours, in which the cytological morphology of the tumour cells and the individual genes mutated in the cancer are more important than size alone. Metastasis is therefore an important, and undermedicated, potential target for cancer therapy [4] , [5] .

Results

Chemotaxis during Tumour Progression One potential explanation for cancer cells becoming metastatic is that they evolve chemotactic competence as the tumours develop [13],[31],[32], and thus move from unsteered to steered migration. We therefore examined the ability of a panel of cell lines isolated from different tumour stages and selected for physiologically appropriate behaviour (Figure 3A) [22]. Surprisingly, all the lines we examined responded chemotactically to serum gradients (Figure 3B). Cells from metastases were more motile than cells from earlier stages (Figure 3C); highly invasive (VGP) cells were slightly more accurate, but not significantly faster than the biologically earlier, RGP cells. Cells from more advanced tumours responded more robustly, but the progression from nonmetastatic to metastatic was not marked by the cells newly acquiring responsiveness—all lines examined were chemotactic enough to spread away from the tumour efficiently in the presence of an appropriate gradient. Several lines of data suggest that genetic and epigenetic changes during progression from RGP to VGP increase cells' ability to survive [33]; our data imply that it is cell survival, rather than chemotactic sensitivity, that defines the difference. The increase in migratory ability could modulate cells' ability to escape from a primary tumour, but our principal conclusion is that melanoma cells from all stages are chemotactic. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Chemotaxis of cells from different melanoma stages. (A) Chemotaxis of a panel of six cell lines from different melanoma stages (RGP, green; VGP, purple; metastatic, red) up a 0%–10% FBS gradient was measured as above (n≥45 cells per cell line). (B) Chemotactic index of cells from different stages. Data from (A) were collated by melanoma stage. Chemotaxis improves as the stage of melanoma progresses, although even the earliest RGP cells show clear chemotaxis. (C) Speeds of cells from different stages. Data from (A) were collated by melanoma stage. Metastatic lines are conspicuously faster (p-values from unpaired t-tests), although again the speed of RGP and VGP cells is still relatively high for non-haematopoietic cells. https://doi.org/10.1371/journal.pbio.1001966.g003

Identifying the Chemoattractant in Serum There are multiple reports of chemotaxis driving metastasis of melanoma and other tumour cells, in particular breast cancer. Published accounts of chemotactic invasion most often describe growth factors as the attractants—for example EGF for solid tumours [34], and EGF, hepatocyte growth factor (HGF), and stem cell factor (SCF)/KitL for melanoma [13]. However these attractants were often identified in transwell chambers, which as earlier discussed are subject to a range of artifacts, in particular false positive. For example, the positive well might promote survival, growth, or adhesion of cells that move randomly across the membrane. Our direct-viewing chambers provide a far more rigorous analysis. We therefore tested a broad range of attractants in our assays. To our surprise, no growth factor acted as an attractant to any measurable degree (Figure 4A); steep or shallow gradients gave no obvious movement upgradient, and no significant chemotactic index towards any growth factor tested (Figure 4B). We therefore conclude that the chemotaxis towards serum we observed was unlikely to be towards growth factors. This does not, of course, demonstrate that melanoma cells are never chemotactic towards growth factors; but it clearly shows the surprising and efficient chemotaxis towards serum observed earlier is mediated by another molecule. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Identification of LPA, rather than growth factors, as the principal attractant in serum. (A) WM239A cells were exposed to gradients of low (light) and high (dark) concentrations of several growth factors and the chemokine SDF-1 in combination with SFM. Spider and Rose plots with Rayleigh tests are shown (n>40 cells for each condition). Concentrations tested were EGF (6.25 and 25 ng/ml), PDGF (25 and 100 ng/ml), HGF (10 and 30 ng/ml), SCF (10 and 100 ng/ml), and SDF-1 (100 and 300 ng/ml). None shows obvious chemotaxis. (B) Quantification of data from (A). Serum gradients promote strong chemotaxis (p<0.0001, unpaired t-test), but gradients of all growth factors tested show no significant chemotactic index (p≥0.40). (C) Growth factors enhance cell speed. Data quantitated from the cells in Figure 3A. Directionless cell speed was measured by totalling the distance moved between time points. EGF and PDGF stimulate cells in serum-free minimal medium to speeds comparable with serum gradients. Single asterisk: Different from SFM alone, p<0.001, unpaired t-test; double asterisk: p<0.0001). (D) LPA and serum drive comparably efficient chemotaxis. WM239A cells were examined in a chamber responding to 0%–10% FBS and 0–1 µM LPA. The spider plot shows similar cellular responses to the two gradients. (E) Quantitative analysis of chemotaxis towards LPA and serum. Chemotactic index was calculated from three experiments including that shown in (D). Cells respond comparably to both conditions. Bars show SEM. https://doi.org/10.1371/journal.pbio.1001966.g004 EGF and PDGF did increase cells' speed (Figure 4C), but they did not provide directional specificity. They therefore acted as chemokines, regulating overall cell behaviour, rather than as chemoattractants that could steer the cells. The striking accuracy of chemotaxis demonstrated by melanoma cells towards serum was more reminiscent of neutrophil chemotaxis towards formyl peptides, or Dictyostelium towards cAMP, which signal through G-protein coupled receptors (GPCRs) rather than growth factor receptors like EGFR and PDGFR. We therefore investigated SDF-1, the ligand for the GPCR CXCR4, which has been associated with poor prognosis and malignancy of melanoma [35]; but again, it was not measurably attractive to cells in our assays (Figure 4B, compare with strong response to serum). However, LPA, another well-known component of serum that signals through GPCRs, was strikingly attractive to melanoma cells. A gradient from 0 to 1 µM LPA across the chamber (consistent with the approximate concentration of LPA in serum; see below) induced chemotaxis almost as effectively as 0%–10% serum (Figure 4D), yielding a comparable chemotactic index (Figure 4E). This was a surprise: LPA is more typically described as an inflammatory mitogen, acting on haematopoietic cells such as macrophages. It appears frequently in the cancer literature, but more often as a mitogen and chemokine for cancer cells, acting via autotaxin, which catalyzes the production of LPA from lysophosphatidylcholine [36]. However in our assays the chemotaxis of melanoma to LPA was again remarkably accurate compared with the weaker chemotaxis typically seen in cancer cells [37].

LPA Is the Dominant Attractant in Serum in 2-D and 3-D Assays To examine whether LPA was the principal attractive component of serum, we assayed chemotaxis in the presence of the antagonist Ki16425, which specifically inhibits binding to LPA receptors 1 and 3 [38]. The effects were again remarkably clear. 10 µM Ki16425 blocked cell spread in our original, density-dependent assay (Movie S4) and chemotaxis towards 10% serum (Figure 5A; Movie S5), reducing the chemotactic index from more than +0.4 to zero (Figure 5B). Ki16425-treated cells were obviously healthy, and moved similarly to untreated cells, with similar track lengths, showing that the treatment was not making the cells nonspecifically sick or non-motile. Knockdown of LPAR1 by siRNA had a similar effect (Figure S2A), showing that LPAR1 is the key receptor for this process, and 10 µM Ki16425 also blocked chemotaxis towards pure LPA (Figure S2B). Again, LPA chemotaxis is not tumour stage-specific; Ki16425 blocked chemotaxis in all cell lines from all stages of cancer progression (Figure 5C). RGP and VGP cell lines were completely inhibited, and the highly motile metastatic lines were substantially inhibited. The residual chemotaxis in the presence of inhibitor could represent either incomplete inhibition by the antagonist, or a small amount of chemotaxis to another agent. From these data, we conclude that LPA is overwhelmingly the dominant chemoattractant in serum for all lines examined. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. LPA responses are essential for serum chemotaxis in 2-D and 3-D assays. (A) LPA receptor antagonist Ki16425 blocks chemotaxis to serum. Chemotaxis of WM239A cells was compared with and without 10 µM Ki16425. Inhibitor-treated cells showed no chemotaxis despite essentially normal random migration. (B) Quantitative analysis of Ki16425 activity. Data from three experiments, including the one in (A). The chemotactic index of inhibitor-treated cells is essentially zero. (C) Melanoma cell lines from all stages chemotaxing up a 10% serum gradient with and without Ki16425. Colours represent melanoma stage. In RGP and VGP cells, chemotaxis is totally blocked, while in metastatic lines it is substantially inhibited. Bars show SEM. (D–E) 3-D organotypic assays. The cell lines WM98-1 and WM1158 are shown ±Ki16425. LPA receptor antagonist greatly inhibits invasion. In (D), invasion index is calculated as the percentage of total cells on the organotypic matrix that invaded beyond ∼30 µm as a ratio of cells on top of the matrix (n>1,000 cells per condition). (E) shows haematoxylin and eosin-stained vertical sections through gels, showing downward invasion of melanoma cells. https://doi.org/10.1371/journal.pbio.1001966.g005 While chamber-based assays are optimized to allow accurate and detailed recording, they provide a 2-D view of a process that more often happens in 3-D tissues in vivo [39]. We therefore examined the role of LPA in a widely used organotypic tumour cell invasion model [40]. In this system melanoma cells are added to the top of a plug of collagen in which fibroblasts are growing, and over time they migrate vertically downwards into the 3-D matrix. During the course of the assay, the collagen plug is set so only its bottom face contacts the medium, at which point malignant melanoma cells invade downwards [41]. We hypothesized that the melanoma cells were driven by a self-generated LPA gradient as in Figure 1B, once fresh LPA could only be supplied from the bottom. This hypothesis is supported by assays in which the collagen plugs remain submerged, and no invasion is seen (Figure S3), further rejecting contact inhibition of migration as a mechanism of invasion. When the gels were treated with Ki16425, the melanoma cells did not invade downwards into the gel (despite comparable numbers of cells at the end, showing no change in growth or survival). Quantitative analysis confirms that Ki16425 strongly inhibited invasion in both cell lines that were invasive in this assay (Figure 5D and 5E). Thus LPA is a dominant steering system for 3-D organotypic assays, as well as for 2-D chamber assays.

Melanoma Cells Break down LPA to Form Outward-Facing Gradients Our earlier data (Figures 1B and 2A, in particular) showed that melanoma cells disperse by depleting a chemoattractant from serum. We therefore tested whether melanoma cells are able to deplete LPA from their surroundings. Full medium with and without serum was incubated with different densities of melanoma cells for different times, then LPA was extracted from the conditioned medium and analyzed by mass spectrometry [42]. This confirms that the melanoma cells effectively break down LPA; the conditioned medium was depleted in a density-dependent manner (Figure 6A) and in a timescale that correlates with the medium conditioning experiments in Figure 2A and 2B. One advantage of using mass spectrometry is the identification of molecular subspecies. The biological activity of LPA is known to vary with its structure [43],[44]. In particular, there is a strong correlation between biological activity and the degree of polyunsaturation, and also acyl chain length [45]. Melanoma cells broke down the biologically active species more rapidly than the others (Figure 6B), ensuring that the most active species also formed the steepest gradients. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Melanoma cells preferentially break down signalling forms of LPA. (A) LPA concentration over 48 hours during conditioning of media, both with and without 10% FBS by melanoma cells (WM239A). FBS conditioned media demonstrates density-dependent depletion of LPA as measured by mass spectrometry. LPA remained negligible throughout 48 hours of serum-free conditioning by the same cells. Representative graph. (B) Analysis of LPA subspecies during melanoma cell conditioning demonstrates bioactive isoforms were depleted more rapidly by melanoma cells in both samples. Two representative graphs are shown to illustrate quantitative variability but qualitative consistency. https://doi.org/10.1371/journal.pbio.1001966.g006

The Role of Growth Factors The results we have obtained conflict with the established dogma that growth factors are primary melanoma chemoattractants [13]. To reconcile these accounts with our data, we examined the role of growth factors during chemotaxis towards LPA. As shown previously (Figure 4C), EGF and (particularly) PDGF increased the basal speed of cells. Gradients of EGF and PDGF, and mixtures of both, enhanced the accuracy of chemotaxis to LPA (Figure 7); LPA, EGF, and PDGF together in serum-free minimal medium were as effective as 10% serum. Most tellingly, however, when cells were presented with LPA and growth factor gradients oriented in opposite directions, they chemotaxed towards the LPA not the growth factors; if anything they migrated towards the LPA with enhanced efficiency (Figure 7B, bottom two lines). Thus when examined in the high levels of detail afforded by our chambers, the growth factors are potentially important accessory factors that increase cell speed and efficiency of chemotaxis, but they do not themselves act as chemoattractants. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 7. Growth factors potentiate LPA chemotaxis. (A) Growth factors enhance cells' response to LPA gradients. Figure shows plots of the WM239A paths chemotaxing in gradients of LPA, LPA+EGF+PDGF, and conflicting gradients of LPA versus EGF+PDGF. (B) Chemotactic indices of cells in (A) and other conditions. Growth factor gradients if anything increase the efficiency of LPA chemotaxis, even when applied in a gradient in the opposite direction. Bars show SEM. https://doi.org/10.1371/journal.pbio.1001966.g007 These results are reminiscent of observations of development in vivo, in which the growth factor SCF promotes migration but not direction of melanoblast migration [46]. It is possible that the melanoma chemotaxis to growth factors observed in other work [13] is due to changes in speed alone, which as discussed earlier can cause a false positive in transwell assays. It has also been shown that growth factors can cause cancer cells to secrete LPA [47], which could also provide an element of indirect chemotaxis in many types of assay.