Optimization of Invasion-Specific Effects of Betulin Derivatives on Prostate Cancer Cells through Lead Development

The anti-invasive and anti-proliferative effects of betulins and abietane derivatives was systematically tested using an organotypic model system of advanced, castration-resistant prostate cancers. A preliminary screen of the initial set of 93 compounds was performed in two-dimensional (2D) growth conditions using non-transformed prostate epithelial cells (EP156T), an androgen-sensitive prostate cancer cell line (LNCaP), and the castration-resistant, highly invasive cell line PC-3. The 25 most promising compounds were all betulin derivatives. These were selected for a focused secondary screen in three-dimensional (3D) growth conditions, with the goal to identify the most effective and specific anti-invasive compounds. Additional sensitivity and cytotoxicity tests were then performed using an extended cell line panel. The effects of these compounds on cell cycle progression, mitosis, proliferation and unspecific cytotoxicity, versus their ability to specifically interfere with cell motility and tumor cell invasion was addressed. To identify potential mechanisms of action and likely compound targets, multiplex profiling of compound effects on a panel of 43 human protein kinases was performed. These target de-convolution studies, combined with the phenotypic analyses of multicellular organoids in 3D models, revealed specific inhibition of AKT signaling linked to effects on the organization of the actin cytoskeleton as the most likely driver of altered cell morphology and motility.Peer reviewe

ShAn: An easy-to-use tool for interactive and integrated variant annotation.

MOTIVATION:Annotation of large amounts of generated sequencing data is a demanding task. Most of the currently available robust annotation tools, like ANNOVAR, are command-line based tools which require a certain degree of programming skills. User-friendly tools for variant annotation of sequencing data with graphical interface are under-represented. RESULTS:We have developed an interactive application, which harnesses the easy usability of R Shiny and combines it with the versatile annotation features of ANNOVAR. This application is easy to use and gives comprehensive annotations for user supplied vcf files using multiples databases. The output table contains the list of variants and their corresponding annotation presented within the graphical interface. In addition, the annotation results are downloadable as text file

Quantification of dynamic morphological drug responses in 3D organotypic cell cultures by automated image analysis

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation

Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention

International audienc

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

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

List of mathematical preliminaries utilized in AMIDA.

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

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