LPA gradients across melanomas <i>in vivo</i>.

<p>(A) TYR::CreER^T2BRAF^V600E/+PTEN^lox/+ mice, a genetically appropriate melanoma model, were treated with tamoxifen as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966-Garcia1" target="_blank">[56]</a>, grown until melanomas spontaneously developed. Dashed box shows the region used for the samples shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio-1001966-g001" target="_blank">Figure 1C</a>. (B) Haematoxylin and eosin-stained biopsies of murine melanomas demonstrating the dispersal of cells from a representative tumour from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio-1001966-g005" target="_blank">Figure 5A</a>, with cells spreading directly away from the tumour. Upper image 2.5× magnification; lower image 20× magnification from dashed box above, showing melanoma cells invading toward the muscle layer (D, dermis; M, muscle layer). (C) Biopsies from mouse melanomas. Several sites in a linear distribution were biopsied using a 6 mm punch biopsy tool within 5 minutes of the mouse being sacrificed and immediately frozen in liquid nitrogen. The positions of biopsies used for LPA measurement are indicated (too few distant samples were obtained for a significant measurement). Bar shows 5 mm. (D) LPA concentration gradients across the margin of a melanoma. Four melanomas were sampled at three sites in a line as shown in (A) (A, tumour body; B, tumour edge; C, skin surrounding tumour). Total LPA per mg tissue was quantified by mass spectrometry after weighing the tissue specimens and extracting the LPA. Outward-directed gradients of LPA were found across the margin of all the melanomas tested. Bars show SEM. (E) Analysis of LPA subspecies. 18∶2-LPA, 20∶4-LPA and 22∶6-LPA show a clearer gradient than 16∶0-LPA, which is though to be less active as a signalling molecule.</p

Density-dependent dispersal of melanoma cells.

<p>(A) Schematic showing the stages of melanoma spread. (B) WM239A metastatic melanoma cells dispersing in uniform medium. 2×10⁴ 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966.s004" target="_blank">Movie S1</a>. 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 2×10⁴ 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. 2×10⁴ 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. 2×10⁴ 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: 2×10⁴ 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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966-MuinonenMartin1" target="_blank">[15]</a>. The cells rapidly migrate towards the well containing serum. Right panel: similar assay with 10% serum in both reservoirs. Panels taken from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966.s006" target="_blank">Movies S3</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966.s004" target="_blank">S1</a>, respectively.</p

Chemotaxis of cells from different melanoma stages.

<p>(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 (<i>n</i>≥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 (<i>p</i>-values from unpaired <i>t</i>-tests), although again the speed of RGP and VGP cells is still relatively high for non-haematopoietic cells.</p

Dispersal is due to a chemoattractant present in serum.

<p>All panels show data from melanoma cells migrating in chemotaxis chambers as described <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001966#pbio.1001966-MuinonenMartin1" target="_blank">[15]</a>. (<b>A–B</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. (<b>C–D</b>) 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. (<b>E</b>) 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 (<i>n</i>>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.</p

Growth factors potentiate LPA chemotaxis.

<p>(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.</p

LPA responses are essential for serum chemotaxis in 2-D and 3-D assays.

<p>(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 (<i>n</i>>1,000 cells per condition). (E) shows haematoxylin and eosin-stained vertical sections through gels, showing downward invasion of melanoma cells.</p

Melanoma cells preferentially break down signalling forms of LPA.

<p>(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.</p