Spheroid assays in different collagen matrices.

<p>Two types of lymphatic endothelial cells, hTERT-HDLECs (a, b) and hMVEC-dly cells (c), were embedded in native collagen (2 mg/ml) (a) or in pepsinized collagen at a low (1,5 mg/ml) (b) or high concentration (2 mg/ml) (c). Cells were cultured in the absence (control) or presence of MMP-inhibitors (RO-28-2653 or MMP2 inhibitor) for 24 h (a, b) or a stimulator (PMA) for 48 h (c). For each assay, the initial spheroid (0 h) and the spheroid at the end of the assay (24 h or 48 h) are shown. Graphs on the right represent the density cell distributions measured around the spheroids. For clarity, the cell density distribution curves for each assay were rendered in three colours (blue, spheroid core; green, edging cells; red, detached cells) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097019#pone-0097019-g001" target="_blank">Figure 1</a>. r_i and r_f correspond to the radius of the initial and final spheroid, respectively. Bars = 500 µm.</p

Global and local measurements of spheroid components upon treatment with a broad-spectrum MMP inhibitor.

<p>Lymphatic hTERT-HDLEC spheroids were embedded in a native collagen matrix with or without (control) an MMP inhibitor (RO-28-2653) for 24 h. The parameters measured are those determined through the assay illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097019#pone-0097019-g002" target="_blank">Figure 2a</a>.</p><p>**P<0,01;</p><p>****P<0,0001;</p><p>*****P<0,00001 (inhibitor versus control).</p

Global and local measurements of spheroid components upon treatment with an MMP2 inhibitor.

<p>Lymphatic hTERT-HDLEC spheroids were embedded in a pepsinized collagen gel with or without (control) an MMP inhibitor (MMP2 inhibitor) for 24 h. The parameters measured are those determined through the assay illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097019#pone-0097019-g002" target="_blank">Figure 2b</a>.</p><p>*P<0,05;</p><p>****P<0,0001 (inhibitor versus control).</p

Description of the spheroid assay and the method of quantification.

<p>(a) Schematic representation of spheroid evolution during cell culture. The initial spheroid is shown on the left (yellow circle) and the different modes of cell sprouting and invasion are depicted on the right panel after 24 h of culture. (b–e) Representative pictures of spheroids after embedding into the collagen matrix (t = 0 h, b) and after 24 h of culture (t = 24 h, d), along with their corresponding binarised images (c, e). (f–h) Decomposition of the binarised image into three components: spheroid core (f), edging cells (g) and detached cells (h). (i) Representation of the whole spheroid and its components: the initial spheroid delineated by a yellow circle, the expanded spheroid core (blue), edging cells (green) and detached cells (red). (j) Illustration of the parameters used for global measurements: convex envelope (green) and total distance of cell invasion starting from the spheroid centre (d₁) or border (d₂). (k) Grid used for local measurements: a circular grid is superimposed on the coloured spheroid representation. (l) Comparison of global and local measurements at t = 0 and t = 24 h. (m) Graph representing the cell density distribution measured from the image. The colours of the curves correspond to the different spheroid components described in the other panels (a, i and k). Bars = 500 µm.</p

Global and local measurements of spheroid components upon treatment with PMA.

<p>Lymphatic hMVEC-dly spheroids were embedded in a pepsinized collagen gel with or without (control) PMA for 48 h. The parameters measured are those determined through the assay illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097019#pone-0097019-g002" target="_blank">Figure 2c</a>.</p><p>**P<0,01;</p><p>***P<0,001;</p><p>****P<0,0001 (inhibitor versus control).</p

Description of 3D spheroid quantification.

<p>(a–d) Representative pictures of immunolabelled spheroids at t = 0 (a) or after 48 h of culture (c) and their corresponding binarised images (b, d). (e–g) Decomposition of the binarised image into the spheroid core (e) and edging cells (f). (g) Representation of the whole spheroid and its components, the spheroid core (blue) and the edging cells (green). (h) Graph representing the cell density distribution measured from the image. Bars = 500 µm.</p

BM-MSC enhance tumor growth and stimulate lymphangiogenesis <i>in vivo</i>.

<p>(A) <i>In vivo</i> bioluminescent signal of tumors developed following injection of 5×10⁴ LLC-Luc cells alone (n = 30) or mixed with 2,5×10⁵ MSC (LLC+MSC) at day 21 (n = 21). The graph corresponds to the quantification of luciferase activity revealing a strong increase of the signal in the LLC+MSC group. (B) BM-MSC enhance lymphatic vessel density in tumors. Sections of tumors induced by injection of LLC-Luc alone (LLC) or with MSC (LLC+MSC) were immunostained with an anti-podoplanin antibody. A computer-assisted quantification of lymphatic vessel density in LLC-Luc tumors (LLC) or in LLC-Luc tumors mixed with BM-MSC (LLC+MSC) is provided on the right. Bar: 100 µm (C) BM-MSC enhance <i>in vivo</i> lymphatic vessel recruitment in sponge implanted in mice ear. Sponges soaked with control medium (CTR; n = 8) or with MSC conditioned medium (MSC CM; n = 7) were implanted in mice ear between skin and cartilage. Lymphatic vessels were identified by LYVE-1 immunolabeling (green) and nuclei were evidenced with Dapi (blue). Bars: 5 mm and 1,5 mm on magnification. * P<0.05. (D). The graphs correspond to LYVE-1 positive lymphatic vessel quantification expressed as (1) the number of vessels plotted as a function of distance to the sponge edge (top graph), and (2) the number of vessels at a distance of 0.3 mm from the edge of the sponge (bottom graph). * P<0.05.</p

VEGF-A secreted by BM-MSC activate LEC.

<p>(A) Western blot analyses of VEGF-A and VEGF-C production on serum-free EBM-2 (CTR) and MSC conditioned medium (MSC CM). (B) VEGFR-2 (top) and VEGFR-3 (bottom) proteins were detected following a phosphorylated tyrosine-containing protein (pY) immunoprecipation (IP) of LEC lysates after cell stimulation with control medium (CTR) or with MSC conditioned medium (MSC CM). Cells treated with VEGF-A (10 ng/ml) or VEGF-C (400 ng/ml) were used as negative and positive controls, respectively. GAPDH western blot was performed on the flowthrough of each sample.</p

BM-MSC stimulate lymphangiogenesis <i>in vitro</i>.

<p>(A) Lymphatic rings were cultured during 5 days alone (CTR) or in presence of BM-MSC spheroids (+MSC spheroids), and during 10 days with control medium (CTR2) or with MSC conditioned medium (MSC CM) prepared as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106976#s2" target="_blank">material and methods</a> section. For quantification, a grid corresponding to successive increments at fixed intervals of explant boundary was used on binarized images and the number of microvessel–grid intersections (N_i) was quantified on binarized images. Quantification was performed at a distance of 0.5 mm and results are expressed as the number of intersections (N_i) plotted as a function of distance (mm) to the lymphatic ring. Bar: 500 µm. * P<0.05. (B, C) MSC conditioned medium significantly stimulates the proliferation and migration of LEC <i>in vitro</i> as compared to control medium. (B) Proliferation rate was measured by a WST-1 and BrdU incorporation assays. ** P<0.01, *** P<0.001. (C) Migration was measured in a Boyden chamber assay. *** P<0.001.</p

VEGF-A is an important factor implicated in LEC stimulation by MSC conditioned medium.

<p>(A, B) The trapping of VEGF-A by the addition of soluble receptors-1 and -2 decreased MSC conditioned medium-induced LEC proliferation, measured by WST-1 assay (A) and migration in a Boyden chamber assay (B). *** P<0.001. (C) Specific inhibition of VEGFR-2 with ZM 323881 10 nM decreased MSC conditioned medium-induce LEC proliferation measured by WST-1 assay. *** P<0.001. (D) Phosphorylation of VEGFR-2 and ERK1/2 analyzed by western blotting on LEC treated or not with soluble VEGF receptors or ZM 323881 10 nM.</p