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

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

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

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