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

Effect of adenosine on LEC migration in scratch test.

<p>HMVEC-dLy were treated during 24 h in medium containing 2% FBS, in the presence of EHNA alone (EHNA, 10 μM) or EHNA with different concentrations of adenosine (AE). Pictures were taken at 0 h, 8 h, 16 h and 24 h after insert removing. Results are expressed as percentage of wound closure (percentage closure) (mean ± SEM). * p<0.05 vs CTRL, ***p<0.001 vs CTRL. Each experiment was performed three times and a representative picture of each condition is shown.</p

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 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

Effect of adenosine on lymphatic vasculature and inflammatory cell recruitment in the liver.

<p>Control adenovirus expressing GFP (CTRL virus) or adenovirus carrying the sequence of 5-nucleotidase (cN-IA virus) were injected in the caudal vein of mice. PBS was used as negative control (PBS). Mice were sacrificed 2 weeks after injection. The efficacy of virus transduction was shown by GFP immunostaining (a) and by cN-IA immunostaining (b)). Liver sections were stained with anti-podoplanin antibodies to detect lymphatic vessels (c), with anti-CD45 antibodies to detect inflammatory cells (d), and with anti-F4/80 antibodies to detect macrophages (e). Data are presented as mean ± SEM (n = 15 for PBS, n = 13 for GFP and n = 12 for cN-IA).**p<0.01, *** p<0.001. Representative pictures are shown.</p

Effect of adenosine on LEC viability and proliferation.

<p>Cell viability (a) and proliferation (b–c) were evaluated in HMVEC-dLy ( = LEC) cultured for 48 h (left panel) or 72 h (right panel) in medium containing 2% FBS (CTRL). Cells were treated with EHNA alone (EHNA 10 μM), EHNA with different concentrations of adenosine (AE), the A2a agonist CGS21680 or the A2b agonist NECA. For cell viability (a), results are expressed as percentage of dead cells (black) and living cells (white). Cell proliferation (b–c) was measured with a CyQANT assay. * p<0.05 vs control (CTRL), *** p<0.001 vs CTRL. Data are expressed as mean ± SEM (n = 3).</p

Effect of adenosine on the migration of LEC in Boyden chamber.

<p>HMVEC-dLy were cultured in medium containing 2% FBS (control condition, CTRL), with or without 10 μM EHNA and 0.1–10 μM adenosine (AE). Cell migration was assessed in a Boyden chamber assay 24 hours after treatment onset. There was no difference on cell migration between all treatments. Results are expressed as percentage of migrating cells (mean ± SEM, n = 3).</p

Effects of adenosine on lymphangiogenesis in the in vivo model of collagen sponge.

<p>Sponges were soaked with PBS as control (CTRL), with VEGF-C (1 ng/ml) as positive control (VEGF-C), with 20 μl CADO (3 ng/ml), a stable analog of adenosine, or with CADO in presence of the A2b antagonist MRS1754 (2.4 mg/ml). Sponges were implanted between the two skin's layers of ear's mice for 3 weeks. Every other day, PBS or MRS1754 were injected in the apex of the ear. Sponge sections were stained with an anti-Lyve-1 antibody to detect lymphatic vessels (green) and Dapi to detect cell nucleus (blue). The graph corresponds to computerized quantification of the surface occupied by lymphatics (vessel area). Data are expressed as mean ± SEM (n = 6). ** p<0.01 vs CTRL.</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