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Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

Muinonen-Martin AJ, Susanto O, Zhang Q, Smethurst E, Faller WJ, Veltman DM, Kalna G, Lindsay C, Bennett DC, Sansom OJ, Herd R, Jones R, Machesky LM, Wakelam MJ, Knecht DA, Insall RH - PLoS Biol. (2014)

Bottom Line: 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.Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

View Article: PubMed Central - PubMed

Affiliation: CRUK Beatson Institute, Glasgow, United Kingdom; York Teaching Hospital NHS Foundation Trust, York, United Kingdom; The Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom.

ABSTRACT
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.

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Related in: MedlinePlus

Dispersal is due to a chemoattractant present in serum.All panels show data from melanoma cells migrating in chemotaxis chambers as described [15]. (A–B) Cells migrate from conditioned medium towards fresh medium. WM1158 cells were randomly attached to a coverslip and assembled in a chamber in 48 hour WM1158 cell conditioned medium. The medium in one chamber was replaced with fresh medium, while the other was left alone. Tracks of individual cells are shown as coloured lines (A). Cells move towards the fresh medium, as shown by the spider plot (B) showing all cell tracks. (C–D) Example images showing WM239A metastatic melanoma cells after 21 hours in serum-free medium (C) and a 0%–10% FBS gradient (D). Coloured paths show centroid tracks from time 0. (E) Quantitative analysis of chemotactic responses. “Spider” plots (large panels), rose plots, mean chemotactic index, and Rayleigh test for directionality are shown for cells in serum-free medium and a 0%–10% FBS gradient (n>100 cells in three independent experiments for both conditions). Spider plots show strong chemotaxis in FBS gradients; in serum-free medium only random movement is seen. Rose plots show overall movement from 6–12 hours; the proportion of total cells in each sector is shown on a log scale, with red lines representing the 95% confidence interval. The majority of cells in the FBS gradient move in the direction of the chemoattractant. Rayleigh tests statistically confirmed this highly significant unimodal directionality. Graphs of chemotactic index were generated from the same data.
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pbio-1001966-g002: Dispersal is due to a chemoattractant present in serum.All panels show data from melanoma cells migrating in chemotaxis chambers as described [15]. (A–B) Cells migrate from conditioned medium towards fresh medium. WM1158 cells were randomly attached to a coverslip and assembled in a chamber in 48 hour WM1158 cell conditioned medium. The medium in one chamber was replaced with fresh medium, while the other was left alone. Tracks of individual cells are shown as coloured lines (A). Cells move towards the fresh medium, as shown by the spider plot (B) showing all cell tracks. (C–D) Example images showing WM239A metastatic melanoma cells after 21 hours in serum-free medium (C) and a 0%–10% FBS gradient (D). Coloured paths show centroid tracks from time 0. (E) Quantitative analysis of chemotactic responses. “Spider” plots (large panels), rose plots, mean chemotactic index, and Rayleigh test for directionality are shown for cells in serum-free medium and a 0%–10% FBS gradient (n>100 cells in three independent experiments for both conditions). Spider plots show strong chemotaxis in FBS gradients; in serum-free medium only random movement is seen. Rose plots show overall movement from 6–12 hours; the proportion of total cells in each sector is shown on a log scale, with red lines representing the 95% confidence interval. The majority of cells in the FBS gradient move in the direction of the chemoattractant. Rayleigh tests statistically confirmed this highly significant unimodal directionality. Graphs of chemotactic index were generated from the same data.

Mentions: We tested this hypothesis using a more traditional chemotaxis assay, in which cells are spread homogeneously over the field at the start of the assay, giving the cells the opportunity to move in any direction [14]. We loaded cells into the chamber in complete medium that had been conditioned by melanoma cells for 48 hours, then replaced the medium in one well with fresh medium containing 10% serum. The cells migrated towards the well containing fresh medium very efficiently (Figure 2A and 2B), showing that an attractant in fresh medium is consumed by the melanoma cells.


Melanoma cells break down LPA to establish local gradients that drive chemotactic dispersal.

Muinonen-Martin AJ, Susanto O, Zhang Q, Smethurst E, Faller WJ, Veltman DM, Kalna G, Lindsay C, Bennett DC, Sansom OJ, Herd R, Jones R, Machesky LM, Wakelam MJ, Knecht DA, Insall RH - PLoS Biol. (2014)

Dispersal is due to a chemoattractant present in serum.All panels show data from melanoma cells migrating in chemotaxis chambers as described [15]. (A–B) Cells migrate from conditioned medium towards fresh medium. WM1158 cells were randomly attached to a coverslip and assembled in a chamber in 48 hour WM1158 cell conditioned medium. The medium in one chamber was replaced with fresh medium, while the other was left alone. Tracks of individual cells are shown as coloured lines (A). Cells move towards the fresh medium, as shown by the spider plot (B) showing all cell tracks. (C–D) Example images showing WM239A metastatic melanoma cells after 21 hours in serum-free medium (C) and a 0%–10% FBS gradient (D). Coloured paths show centroid tracks from time 0. (E) Quantitative analysis of chemotactic responses. “Spider” plots (large panels), rose plots, mean chemotactic index, and Rayleigh test for directionality are shown for cells in serum-free medium and a 0%–10% FBS gradient (n>100 cells in three independent experiments for both conditions). Spider plots show strong chemotaxis in FBS gradients; in serum-free medium only random movement is seen. Rose plots show overall movement from 6–12 hours; the proportion of total cells in each sector is shown on a log scale, with red lines representing the 95% confidence interval. The majority of cells in the FBS gradient move in the direction of the chemoattractant. Rayleigh tests statistically confirmed this highly significant unimodal directionality. Graphs of chemotactic index were generated from the same data.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4196730&req=5

pbio-1001966-g002: Dispersal is due to a chemoattractant present in serum.All panels show data from melanoma cells migrating in chemotaxis chambers as described [15]. (A–B) Cells migrate from conditioned medium towards fresh medium. WM1158 cells were randomly attached to a coverslip and assembled in a chamber in 48 hour WM1158 cell conditioned medium. The medium in one chamber was replaced with fresh medium, while the other was left alone. Tracks of individual cells are shown as coloured lines (A). Cells move towards the fresh medium, as shown by the spider plot (B) showing all cell tracks. (C–D) Example images showing WM239A metastatic melanoma cells after 21 hours in serum-free medium (C) and a 0%–10% FBS gradient (D). Coloured paths show centroid tracks from time 0. (E) Quantitative analysis of chemotactic responses. “Spider” plots (large panels), rose plots, mean chemotactic index, and Rayleigh test for directionality are shown for cells in serum-free medium and a 0%–10% FBS gradient (n>100 cells in three independent experiments for both conditions). Spider plots show strong chemotaxis in FBS gradients; in serum-free medium only random movement is seen. Rose plots show overall movement from 6–12 hours; the proportion of total cells in each sector is shown on a log scale, with red lines representing the 95% confidence interval. The majority of cells in the FBS gradient move in the direction of the chemoattractant. Rayleigh tests statistically confirmed this highly significant unimodal directionality. Graphs of chemotactic index were generated from the same data.
Mentions: We tested this hypothesis using a more traditional chemotaxis assay, in which cells are spread homogeneously over the field at the start of the assay, giving the cells the opportunity to move in any direction [14]. We loaded cells into the chamber in complete medium that had been conditioned by melanoma cells for 48 hours, then replaced the medium in one well with fresh medium containing 10% serum. The cells migrated towards the well containing fresh medium very efficiently (Figure 2A and 2B), showing that an attractant in fresh medium is consumed by the melanoma cells.

Bottom Line: 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.Thus the stimulus that drives cell dispersal is not the presence of LPA itself, but the self-generated, outward-directed gradient.

View Article: PubMed Central - PubMed

Affiliation: CRUK Beatson Institute, Glasgow, United Kingdom; York Teaching Hospital NHS Foundation Trust, York, United Kingdom; The Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus