Our compound screening concept is based on a simple cell culture platform optimised for 3D spheroid cultures complemented with an easy to use proprietary image analysis program and data analysis tools.

<p>(A) ibidi Angiogenesis μ-slides and μ-plates have a unique well-in-a-well design that facilitates 3D cell culturing between two layers of extracellular matrix on a very narrow focal plane. (B) Time and operation schedule for a typical compound screen including all the major steps from cell seeding to image analysis and visualisation of morphometric responses.</p

Exemplary screen based on the PC-3 spontaneous invasive transformation model.

<p>PC-3 spheroids were treated with 19 compounds mainly targeting integrity, function and organization of the actin cytoskeleton. 172–424 multicellular structures for each treatment were analysed with AMIDA program. (A) A morphometric heatmap showing standardized differences in medians between the treatments and the control for 15 morphological parameters and all 19 compound treatments. Morphological responses clustered into three functional groups. Increasing cytotoxicity, measured by the AreaRatioR parameter – based on presence of dead cells stained with ethidium homodimer - is indicated by the red gradient arrow. (B) Correlation map (nonparametric Spearman) indicating the similarity (positive correlation, red) or dissimilarity (negative correlation, blue) for 21 of AMIDAs morphometric parameters. (C) Bonferroni-corrected and Mann-Whitney U-test filtered morphometric heatmap (threshold p>0.05) focusing on four selected, most informative parameters (AppIndex, AreaRatioR, Roundness, Area). The graph highlights compounds causing mainly growth-inhibition and cytotoxicity (group II), and those that enhance spheroid symmetry and reduce number of invasive protrusions (group I). (D) The image panel shows representative, segmented PC-3 spheroids for groups I and II, compared to DMSO and paclitaxel controls, after six days of drug treatment.</p

List of morphological parameters implemented in AMIDA.

<p><i>*not included in the result file</i>.</p><p><i>**Computed for both channels {R,G} separately</i>.</p

List of compounds used in exemplary screens.

<p>List of compounds used in exemplary screens.</p

Validation of morphological responses with 9 additional prostate and 3 breast cancer cell lines.

<p>The heatmaps illustrate changes in spheroid growth (A: Area) and general symmetry (B: Roundness) in response to the 19 compound treatments (5031–16415 multicellular structures analysed for each cell line). (A) CCG-1423, KH7, latrunculin A and narciclasine are preferentially cytotoxic and/or antiproliferative compounds across all cell lines, as highlighted by red boxes. Paclitaxel, at a concentration of 5 nM, shows partial cytotoxic/antiproliferative effects only in some of the cell lines. (B) Effects of mainly anti-invasive compounds IPA3 or NSC23766 were reproducible in many of the spontaneously invasive (or branching) cell lines PC-3, PC-3M Pro4, and RWPE-1. (C) Images segmented and analysed with AMIDA. Effective anti-invasive functions of the compounds IPA3, BPIPP and NSC23766 against the most aggressive, motile and invasive cell lines PC-3M, ALVA31, MDA-MB-231 (both SA and parental ATCC), and RWPE1. The extremely invasive PC3-derivative ALVA31 was not affected, however. (D) Blebbistatin and Y-27632 show invasion-inducing function in spheroids formed by the LNCaP and DU145 lines which typically form round spheroids and lack invasive properties.</p

Evaluation of key parameters analysed by AMIDA.

<p>(A: left panel) Six representative PC-3 spheroids, all treated with different compounds in order to manipulate the morphology, stained for viable cells (Calcein AM) and imaged with spinning disk confocal microscope (5x objective). (A: right panel) The same spheroids segmented with AMIDA. The table in A shows numerical values appointed by AMIDA for selected morphological features. (B) Representative panel of PC-3 spheroids with red dots added by image manipulation in certain number and distribution to exemplify the power and preciseness of RGB functions (B: table).</p

List of mathematical preliminaries utilized in AMIDA.

<p>List of mathematical preliminaries utilized in AMIDA.</p

Histograms showing the distribution of morphological response data in the exemplary screen.

<p>The data is shown for three key parameters, Area (representing spheroid size), Roundness and AppIndex (representing symmetry), and for three experimental compounds each one representing one of the response groups (DMSO: control, NF023: no response, EHT-1864: growth inhibition, gallein: anti-invasive).</p

Primary 3D screen.

<p>PC-3 cells were cultured in 3D Matrigel ECM for 4 days and treated for 6 days with 25 betulin derivatives (from 2D high throughput screens), DMSO/vehicle control, and three reference compounds. A) Representative maximum intensity projections of confocal microscope stack images for selected compound treatments at 300 nM concentration (5× objective, scale 100 μm). B) Three graphs showing the relative impact on three morphometric parameters for 7 betulin derivatives, DMSO control, and one control compound (paclitaxel). Data scaling: displays the relative difference between median of Area/Complexity/Area Ratio, to DMSO control. Paclitaxel treatments and DMSO controls have been assigned values of -100 and 0, respectively. C) Hierarchical clustering was done using three morphological parameters derived from PC3 organoids: spheroid size (area), complexity and the number of dead cells. D) Wound healing curves of the two betulin derivatives <b>4</b> and <b>20</b>, highlighting the 50% cut-off level (orange dashed line).</p

Secondary 3D screens.

<p>Experimental betulin derivatives and control compounds were tested across a panel of prostate cancer lines (LNCaP, LAPC-4) and non-transformed, prostate epithelial cells (EP156T) in 3D culture. <b>A</b>) The dendrogram is based on three main morphological parameters and combines all data from primary and secondary 3D screens. Effective compound treatments are indicated in red and yellow, yellow being the most invasion-specific. <b>B</b>) Boxplots showing the impact of selected compounds on spheroid size (Area). Data scaling: as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126111#pone.0126111.g004" target="_blank">Fig 4</a><b>C</b>) Effects of all betulin derivatives and control compounds on cell death (number of dead cells).</p