A novel 3D high-content assay identifies compounds that prevent fibroblast invasion into tissue surrogates.

Invasion processes underlie or accompany several pathological processes but only a limited number of high-throughput capable phenotypic models exist to test anti-invasive compounds in vitro. We here evaluated 3D co-cultures as a high-content phenotypic screening system for fibrotic invasive processes. 3D multicellular spheroids were used as living tissue surrogates in co-culture with fluorescently labeled lung fibroblasts to monitor invasion processes by automated microscopy. This setup was used to screen a compound library containing 480 known bioactive substances. Identified hits prevented fibroblast invasion and could be subdivided into two hit classes. First, Prostaglandins were shown to prevent fibroblast invasion, most likely mediated by the prostaglandin EP2 receptor and generation of cAMP. Additionally, Rho-associated protein kinase (ROCK) inhibitors prevented fibroblast invasion, possibly by inactivation of myosin II. Importantly, both Prostaglandins and ROCK inhibitors are potential treatment options shown to be effective in in vitro and in vivo models of fibrotic diseases. This validates the presented novel phenotypic screening approach for the evaluation of potential inhibitors and the identification of novel compounds with activity in diseases that are associated with fibroblast invasion.

3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.

Cancer cells in poorly vascularized tumor regions need to adapt to an unfavorable metabolic microenvironment. As distance from supplying blood vessels increases, oxygen and nutrient concentrations decrease and cancer cells react by stopping cell cycle progression and becoming dormant. As cytostatic drugs mainly target proliferating cells, cancer cell dormancy is considered as a major resistance mechanism to this class of anti-cancer drugs. Therefore, substances that target cancer cells in poorly vascularized tumor regions have the potential to enhance cytostatic-based chemotherapy of solid tumors. With three-dimensional growth conditions, multicellular tumor spheroids (MCTS) reproduce several parameters of the tumor microenvironment, including oxygen and nutrient gradients as well as the development of dormant tumor regions. We here report the setup of a 3D cell culture compatible high-content screening system and the identification of nine substances from two commercially available drug libraries that specifically target cells in inner MCTS core regions, while cells in outer MCTS regions or in 2D cell culture remain unaffected. We elucidated the mode of action of the identified compounds as inhibitors of the respiratory chain and show that induction of cell death in inner MCTS core regions critically depends on extracellular glucose concentrations. Finally, combinational treatment with cytostatics showed increased induction of cell death in MCTS. The data presented here shows for the first time a high-content based screening setup on 3D tumor spheroids for the identification of substances that specifically induce cell death in inner tumor spheroid core regions. This validates the approach to use 3D cell culture screening systems to identify substances that would not be detectable by 2D based screening in otherwise similar culture conditions.

Subgroup-elimination transcriptomics identifies signaling proteins that define subclasses of TRPV1-positive neurons and a novel paracrine circuit.

Normal and painful stimuli are detected by specialized subgroups of peripheral sensory neurons. The understanding of the functional differences of each neuronal subgroup would be strongly enhanced by knowledge of the respective subgroup transcriptome. The separation of the subgroup of interest, however, has proven challenging as they can hardly be enriched. Instead of enriching, we now rapidly eliminated the subgroup of neurons expressing the heat-gated cation channel TRPV1 from dissociated rat sensory ganglia. Elimination was accomplished by brief treatment with TRPV1 agonists followed by the removal of compromised TRPV1(+) neurons using density centrifugation. By differential microarray and sequencing (RNA-Seq) based expression profiling we compared the transcriptome of all cells within sensory ganglia versus the same cells lacking TRPV1 expressing neurons, which revealed 240 differentially expressed genes (adj. p<0.05, fold-change>1.5). Corroborating the specificity of the approach, many of these genes have been reported to be involved in noxious heat or pain sensitization. Beyond the expected enrichment of ion channels, we found the TRPV1 transcriptome to be enriched for GPCRs and other signaling proteins involved in adenosine, calcium, and phosphatidylinositol signaling. Quantitative population analysis using a recent High Content Screening (HCS) microscopy approach identified substantial heterogeneity of expressed target proteins even within TRPV1-positive neurons. Signaling components defined distinct further subgroups within the population of TRPV1-positive neurons. Analysis of one such signaling system showed that the pain sensitizing prostaglandin PGD2 activates DP1 receptors expressed predominantly on TRPV1(+) neurons. In contrast, we found the PGD2 producing prostaglandin D synthase to be expressed exclusively in myelinated large-diameter neurons lacking TRPV1, which suggests a novel paracrine neuron-neuron communication. Thus, subgroup analysis based on the elimination rather than enrichment of the subgroup of interest revealed proteins that define subclasses of TRPV1-positive neurons and suggests a novel paracrine circuit.